Recombinant virus and preparations thereof

ABSTRACT

The present invention generally relates to methods and compositions used delivery of gene editing compositions including transcriptional effectors with parvovirus and preferred methods for making same.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation-in-part of international patent application Serial No. PCT/US2014/030394, filed Mar. 17, 2014, and published as PCT Publication No. WO2014/145599 on Sep. 18, 2014 and which claims priority to U.S. patent application Ser. No. 14/213,991 filed on Mar. 14, 2014 which claims priority to U.S. Provisional Application 61/799,800 filed on Mar. 15, 2013. Reference is made to US applications having Broad reference BI-2011/008 to US Provisional Application Nos. 61/736,527 filed Dec. 12, 2012; 61/748,427 filed Jan. 2, 2013; 61/757,972 filed Jan. 29, 2013, 61/768,959, filed Feb. 25, 2013 and 61/791,409 filed Mar. 15, 2013, titled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION; Broad reference BI-2011/020 to US Provisional Application Nos. 61/675,778 filed Jul. 25, 2012; 61/721,283 filed Nov. 1, 2012: 61/726,465 filed Dec. 12, 2012 and 61/794,458 filed Mar. 15, 2013, tided. INDUCIBLE DNA BINDING PROTEINS AND GENOME PERTURBATION TOOLS AND APPLICATIONS THEREOF; Broad reference BI-2011/021 to U.S. Provisional Application No. 61/565,171 filed Nov. 30, 2011 and U.S. application Ser. No. 13/554,922 filed Jul. 30, 2012 and Ser. No. 13/604,945 filed Sep. 6, 2012, titled NUCLEOTIDE-SPECIFIC RECOGNITION SEQUENCES FOR DESIGNER TAL EFFECTORS and Broad references BI-2013/003 and BI-2013/004 to U.S. Provisional Application No. 61/836,123 filed on Jun. 17, 2013 and U.S. Provisional Application Nos. 61/758,468; 61/769,046; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130 each entitled ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION, filed on Jan. 30, 2013; Feb. 25, 2013; Mar. 15, 2013; Mar. 28, 2013; Apr. 20, 2013; May 6, 2013 and May 28, 2013 respectively.

The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

FEDERAL FUNDING LEGEND

This invention was made with government support under grant numbers NS073124 and MH100706 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to methods for preparation of viral vector and methods and compositions for advantageous delivery of nucleic acid molecule(s) for expression of Transcription Activation Like Effector (TALE) and nucleic acid molecule(s) for expression of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, or nucleic acid molecule(s) for expression of a light-inducible transcriptional effector (LITE), or a cassette or plurality of cassette comprising or consisting essentially of a promoter and exogenous nucleic acid molecule encoding same particularly for gene editing in a eukaryote cell. TALEs, LITEs and CRISPRs expressed via a recombinant construct, e.g., an AAV, can advantageously provide activator, repressor or nuclease activity in vivo, in vitro or ex vivo.

The method of the invention can provide a readily accessible, reproducible aliquot of recombinant construct that can be used for testing, e.g., testing whether construction of the recombinant construct was successful, or whether the recombinant construct expresses the exogenous DNA in an amount that may be sufficient for an intended use and/or for a duration that may be sufficient for an intended use, i.e., for screening, such as high throughput screening. And hence the invention relates to a method that may advantageously be for screening or high throughput screening, wherein the method additionally comprises or consists essentially of contacting the aliquot with cells and determining whether the exogenous DNA is expressed in an amount and/or duration sufficient for an intended use.

BACKGROUND OF THE INVENTION

Normal gene expression is a dynamic process with carefully orchestrated temporal and spatial components, the precision of which are necessary for normal development, homeostasis, and advancement of the organism. In turn, the dysregulation of required gene expression patterns, either by increased, decreased, or altered function of a gene or set of genes, has been linked to a wide array of pathologies. Technologies capable of modulating gene expression in a spatiotemporally precise fashion will enable the elucidation of the genetic cues responsible for normal biological processes and disease mechanisms. To address this technological need, Applicants developed molecular tools that may regulate gene expression.

There is an evident need for methods and compositions that allow for efficient and precise spatial and temporal control of a genomic locus of interest. These methods and compositions may provide for the regulation and modulation of genomic expression both in vivo and in vitro as well as provide for novel treatment methods for a number of disease pathologies.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus which is endogenous to the human population. Although capable of productive infection in cells from a variety of species, AAV is a dependovirus, requiring helper functions from either adenovirus, herpesvirus or a poxvirus such as vaccinia virus for its own replication. In the absence of helper functions from any of these helper viruses, AAV will infect cells, uncoat in the nucleus, and integrate its genome into the host chromosome, but will not replicate or produce new viral particles. There are at least 12 recognized AAV serotypes, There are recombinant AAVs. A recombinant AAV can accommodate approximately 4300 bases of exogenous DNA, and AAVs having a hybrid or mosaic capsid have been produced.

The genome of AAV has been cloned into bacterial plasmids and is well characterized. The viral genome consists of 4682 bases which include two terminal repeats of 145 bases each. These terminal repeats serve as origins of DNA replication for the virus. Some investigators have also proposed that they have enhancer functions. The rest of the genome is divided into two functional domains. The left portion of the genome codes for the rep functions which regulate viral DNA replication and vital gene expression. The right side of the vital genome contains the cap genes that encode the structural capsid proteins VP1, VP2 and VP3. The proteins encoded by both the rep and cap genes function in trans during productive AAV replication.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY OF THE INVENTION

The present invention particularly relates to methods for preparation of viral vector and methods and compositions for advantageous delivery of Transcription Activation Like Effector (TALE) and nucleic acid molecule(s) for expression or a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, or a cassette or plurality of cassette comprising or consisting essentially of a promoter and exogenous nucleic acid molecule encoding same particularly for gene editing in a eukaryote cell.

The present invention encompasses nucleic acid encoding the polypeptides of the present invention. The nucleic acid may comprise a promoter, advantageously human Synapsin I promoter (hSyn). In one embodiment, the nucleic acid is packaged into a viral vector. In some embodiments, the nucleic acid is packaged into a parvovirus-based vector. In some embodiments, the nucleic acid is packaged into an adeno associated viral vector (AAV).

The invention further relates to methods of treatment or therapy that encompass the methods and compositions described herein.

As discussed herein, the present invention generally relates to recombinant parvovirus (Group II viruses according to the Baltimore classification; e.g., Parvovirus B19, e.g. Dependovirus (e.g. Adeno-Associated Virus or AAV), Erythrovirus (e.g. Parvovirus B19) or Bocavirus), advantageously AAV. AAV is a prototypical Dependovirus, The invention will be discussed with regard to advantageous AAV embodiments with it understood that the invention comprehends any of “parvovirus”, “Parvovirus B19”. “Dependovirus”, “Erythrovirus” or “Bocavirus” or species or serotypes of any of the foregoing in place of “AAV” in discussion herein. It is also understood that “AAV”, unless specified as being a particular serotype or specified as having a particular capsid can be any of the herein identified AAVs.

There is a need for TALEs and LITEs to be expressed via a recombinant construct, e.g., an AAV, e.g., to provide activator, repressor or nuclease activity in vivo, in vitro or ex vivo.

There is a need for expression of a CRISPR system via a recombinant construct, e.g., an AAV, e.g., to provide knockdown in vivo, in vitro or ex vivo by the CRISPR introducing a spacer, which inhibits a target gene.

As traditional AAV or rAAV production requires a laborious production and purification process from cells, e.g., HEK-293FT cells, and this can make testing many constructs in parallel impractical. There is a need for a simple yet highly effective method of preparing AAV or rAAV, including testing or screening thereof, e.g., high throughput screening, and methods of using the resulting AAV or rAAV to integrate into the genome of cells otherwise difficult to infect, such as non-dividing cells, although AAV is able to infect both dividing and quiescent cells. In one aspect neuronal cells are targetted e.g., via neuronal transduction. Means for neuronal transduction also can be ascertained via Mason et al, “Comparison of AAV Serotypes for Gene Delivery to Dorsal Root Ganglion Neurons,” Mol Ther. 2010 April; 18(4): 715-724 (2010 Feb. 23). All types of AAV and other Dependovirus are known to infect multiple diverse tissue types, and various AAV serotypes are known to have natural tropism to different tissues depending on their capsid proteins. Target tissues include, but are not limited to, e.g., brain, neurons, liver, eye, cardiac, muscle, and even cancer. See, e.g., Alam et al., Mol Cancer. 2011 Aug. 9; 10:97; Bartel et al. Gene Ther. 2012 June; 19(6):694-700.

There is also a need for a readily accessible, reproducible aliquot of recombinant construct that can be used for testing whether construction of the recombinant construct was successful, or whether the recombinant construct expresses the exogenous DNA in an amount that may be sufficient for an intended use and/or for a duration that may be sufficient for an intended use, i.e., for screening, such as high throughput screening, for therapeutic uses such as gene therapy, and targeting a broad range of tissues, whether of dividing or quiescent cells. Thus, there is a need for methods of the invention including those that may advantageously be for screening or high throughput screening, wherein the method includes or consists essentially of contacting the aliquot with cells and determining whether the exogenous DNA is expressed in an amount and/or duration sufficient for an intended use, e.g., gene therapy, genetic engineering or screening.

AAV is considered an ideal candidate for use as a transducing vector. Such AAV transducing vectors can comprise sufficient cis-acting functions to replicate in the presence of adenovirus or herpesvirus or poxvirus (e.g., vaccinia virus) helper functions provided in trans. Recombinant AAV (rAAV) can be used to carry exogenous genes into cells of a variety of lineages. In these vectors, the AAV cap and/or rep genes are deleted from the viral genome and replaced with a DNA segment of choice. Current AAV vectors may accommodate up to 4300 bases of inserted DNA.

There are a number of ways to produce rAAV, and the invention provides rAAV compositions and methods for preparing rAAV. For example, plasmid(s) containing or consisting essentially of the desired viral construct are transfected into AAV-infected cells. In addition, a second or additional helper plasmid is cotransfected into these cells to provide the AAV rep and/or cap genes which are obligatory for replication and packaging of the recombinant viral construct. Under these conditions, the rep and/or cap proteins of AAV act in trans to stimulate replication and packaging of the rAAV construct. Two to three days after transfection, rAAV is harvested. Traditionally rAAV is harvested from the cells along with adenovirus. The contaminating adenovirus is then inactivated by heat treatment. In the instant invention, rAAV is advantageously harvested not from the cells themselves, but from cell supernatant. Accordingly, in an initial aspect the invention provides for preparing rAAV, and in addition to the foregoing, rAAV can be prepared by one or more methods that comprise or consist essentially of,

-   -   infecting susceptible cells with a rAAV containing exogenous DNA         including DNA for expression, and helper virus (e.g.,         adenovirus, herpesvirus, poxvirus such as vaccinia virus)         wherein the rAAV lacks functioning cap and/or rep (and the         helper virus (e.g., adenovirus, herpesvirus, poxvirus such as         vaccinia virus) provides the cap and/or rev function that the         rAAV lacks); or     -   infecting susceptible cells with a rAAV containing exogenous DNA         including DNA for expression, wherein the recombinant construct         lacks functioning cap and/or rep, and transfecting said cells         with a plasmid supplying cap and/or rep function that the rAAV         lacks; or     -   infecting susceptible cells with a rAAV containing exogenous DNA         including DNA for expression, wherein the recombinant construct         lacks functioning cap and/or rep, wherein said cells supply cap         and/or rep function that the recombinant construct lacks; or     -   transfecting the susceptible cells with an AAV lacking         functioning cap and/or rep and plasmids for inserting exogenous         DNA into the recombinant construct so that the exogenous DNA is         expressed by the recombinant construct and for supplying rep         and/or cap functions whereby transfection results in an rAAV         containing the exogenous DNA including DNA for expression that         lacks functioning cap and/or rep.     -   In addition to methods for preparing rAAV, the invention         provides methods for using such recombinant constructs, and         compositions or preparations of such recombinant constructs,         including without limitation compositions or preparations         resulting from a method for obtaining and optionally storing a         sample containing a set amount of rAAV; and, this method can         further optionally include testing the rAAV.

The method advantageously may comprise or consist essentially of, and hence the invention pertains to a method for obtaining and optionally storing a sample containing a set amount of rAAV comprising or consisting essentially of:

-   -   preparing the rAAV as herein described, e.g.,         -   plasmid(s) containing or consisting essentially of the             desired viral construct are transfected into AAV-infected             cells along with another helper plasmid that provide the AAV             rep and/or cap genes which are obligatory for replication             and packaging of the recombinant viral construct; or         -   infecting susceptible cells with a rAAV containing exogenous             DNA including DNA for expression, and helper virus (e.g.,             adenovirus, herpesvirus, poxvirus such as vaccinia virus)             wherein the rAAV lacks functioning cap and/or rep (and the             helper virus (e.g., adenovirus, herpesvirus, poxvirus such             as vaccinia virus) provides the cap and/or rev function that             the rAAV lacks); or         -   infecting susceptible cells with a rAAV containing exogenous             DNA including DNA for expression, wherein the recombinant             construct lacks functioning cap and/or rep, and transfecting             said cells with a plasmid supplying cap and/or rep function             that the rAAV lacks; or         -   infecting susceptible cells with a rAAV containing exogenous             DNA including DNA for expression, wherein the recombinant             construct lacks functioning cap and/or rep, wherein said             cells supply cap and/or rep function that the recombinant             lacks; or         -   transfecting the susceptible cells with an AAV lacking             functioning cap and/or rep and plasmids for inserting             exogenous DNA into the recombinant construct so that the             exogenous DNA is expressed by the recombinant construct and             for supplying rep and/or cap functions whereby transfection             results in an rAAV containing the exogenous DNA including             DNA for expression that lacks functioning cap and/or rep;             and     -   incubating the infected or transfected cells, whereby there         results infected or transfected cells and supernatant containing         the rAAV lacking functioning cap and/or rep;     -   after incubating, extracting an aliquot from the supernatant;     -   filtering the aliquot, whereby the filtered aliquot contains and         the method obtains a sample containing set amount of the rAAV         relative to the type and amount of susceptible cells infected or         transfected; and     -   optionally freezing the filtered aliquot, whereby the method         optionally includes storing a sample containing set amount of         the rAAV relative to the type and amount of susceptible cells         infected or transfected.

The rAAV can be from an AAV as herein described, and advantageously can be an rAAV1, rAAV2, AAV5 or rAAV having a hybrid capsid which may comprise AAV1, AAV2, AAV5 or any combination thereof. One can select the AAV of the rAAV with regard to the cells to be targeted by the rAAV; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid or capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue.

The susceptible cells are advantageously 293FT cells. The method advantageously includes or consists essentially of freezing (e.g., about −80° C.) the filtered aliquot. A secretion enhancer (e.g., polyethylenimine (PEI)) may be added to the cells before, during or after and within the incubating. The incubating can be typically up to 48 or 72 hours. 2×10⁵ cells are advantageously transfected or infected, especially when the cells are 293FT cells. The filtered aliquot advantageously has a volume of 250 μL.

When the cells are 293FT cells and 2×10⁵ cells are advantageously transfected or infected, the rAAV concentration in the filtered 250 μL, aliquot is approximately 5.6+/−0.24×10⁵. When cells other than 293FT are used, there should be a linear relationship with regard to the amount of rAAV in the supernatant, aliquot and filtered aliquot. Thus, from 2×10⁵ 293 FT cells obtaining the rAAV concentration in the filtered 250 μL aliquot of approximately 5.6+/−0.24×10⁵, the skilled person can transfect the same number of other cells and measure the viral output (e.g., via qPCR) and ascertain the linear relationship amongst cells. Other cells that can be used in the practice of the invention and the relative infectivity of certain AAV serotypes in vitro as to these cells (see Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) are as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 100 2.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 100 2.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.2 1.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 333 50 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.0 0.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.5 0.1 HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 ND ND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100 ND ND 333 3333 ND ND

The invention provides rAAV that contains or consists essentially of an exogenous nucleic acid molecule encoding a transcriptional effector such as a Transcription Activation Like Effector (TALE) and nucleic acid molecule(s) for expression or a cassette comprising or consisting essentially of a promoter and a nucleic acid molecule encoding a transcriptional effector such as a TALE.

The invention provides rAAV that contains or consists essentially of an exogenous nucleic acid molecule encoding an inducible transcriptional effector such as a light-inducible transcriptional effector (LITE) and nucleic acid molecule(s) for expression or a cassette comprising or consisting essentially of a promoter and a nucleic acid molecule encoding an inducible transcriptional effector such as a LITE.

The invention provides rAAV that contains or consists essentially of an exogenous nucleic acid molecule encoding a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, e.g., a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase proteins), e.g., Cas9 and a terminator, and a two, or more, advantageously up to the packaging size limit of the vector, e.g., in total (including the first cassette) five, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . . Promoter-gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector), or two or more individual rAAVs, each containing one or more than one cassette of a CRISPR system, e.g., a first rAAV containing the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding Cas, e.g., Cas9 and a terminator, and a second rAAV containing a plurality, four, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator . . . Promoter-gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector).

As rAAV is a DNA virus, the nucleic acid molecules in the herein discussion are advantageously DNA.

The invention also provides a readily accessible, reproducible aliquot of rAAV that can be used for testing, e.g., testing whether construction of the rAAV was successful, or whether the rAAV expresses the exogenous DNA in an amount that may be sufficient for an intended use and/or for a duration that may be sufficient for an intended use, i.e., for screening, such as high throughput screening.

Hence, the invention provides a method for screening or high throughput screening, wherein the method comprises or consists essentially of preparing the filtered aliquot or the stored filtered aliquot as herein described, if necessary, thawing the stored filtered aliquot, contacting the filtered aliquot with cells and determining whether the exogenous DNA is expressed in an amount and/or duration sufficient for an intended use. The contacting with cells can be transducing said cells (e.g., contacting can take 5-6 days with observation whereby suitable levels of rAAV expression are reached). For instance, the rAAV can express a TALE and the contacting can include detecting nuclease, activator or repressor activity. The rAAV can express an inducible transcriptional effector such as a LITE, and the contacting can include inducing gene expression or subjecting the contacted cells to a suitable stimulus, and if detecting whether transcriptional effector has been induced, e.g., via detecting a color change. The rAAV can express a CRISPR system, and the contacting can include detecting gene knockdown or other effects of the CRISPR system.

The invention further provides advantageous methods of AAV or rAAV production. In one aspect, as further described in the Examples herein, the invention encompasses AAV supernatant production. The methods of the invention described herein comprehend varying the DNA ratios of the vectors used, e.g. the ratios of vector of interest plasmid: AAV serotype plasmid: pHelper plasmid may be varied. In a preferred embodiment of the invention, this value may be 1:1.7:2 for AAV supernatant production down to 24-well scale. In another preferred embodiment of the invention, this value may be 1:2:1 for a 96-well format.

The invention also comprehends the scaling up of the AAV supernatant production to higher throughput formats. Aspects of the invention may be carried out in a 15 cm dish. In a further embodiment, aspects of the invention comprehend scaling up from a 15 cm dish to 96-well plates for production. In another aspect, the invention also encompasses scaling up which includes but is not limited to 384-well plates or 1536-well plates. In a further embodiment, the invention also comprehends a microfluidic device capable of maintaining cell cultures in individual chambers. In a preferred embodiment, the AAV supernatant produced in the methods of the invention may be produced at the same scale as it may be applied.

The invention provides for methods of filtration or purification of the supernatant containing AAV generated in the methods described herein. Methods of filtration or purification may include but are not limited to the use of filters or centrifugation. In one aspect of the invention, filtration with specific pore size filters may be employed to remove any potential 293FT cells and large cell debris. In a preferred embodiment, a 22 micron or 45 micron pore size low protein binding filter may be used. When filtration is utilized the flow-through is harvested and subsequently used. In another aspect of the invention, centrifugation may be employed to pellet cells and cell debris. In a preferred embodiment, centrifugation at speeds in the range of 200 g for 20 min to 6000 g for 1-10 min may be utilized. When centrifugation is utilized the supernatant is collected and subsequently used. In a further embodiment of the invention, these steps may be followed by subsequent purification steps when more stringent purification is desired. In a preferred embodiment a sequence of molecular weight cutoff filters (e.g. amicon filters, Millipore) may be used.

The invention also provides for methods of AAV supernatant production which do not use fetal bovine serum (FBS). In a preferred embodiment, the culture medium used to support AAV producing 293FT cells may be replaced with a chemically-defined serum-free medium. e.g. Pro293a.

The invention also provides for AAV supernatant production methods being used to generate functional pooled AAV supernatant. Furthermore, the invention also provides for multiple supernatant AAV batches being harvested from a single AAV producing 293FT culture.

Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

The invention further also provides other recombinant constructs, compositions, preparations, and methods described herein.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

FIG. 1 shows a schematic indicating the need for spatial and temporal precision.

FIG. 2 shows transcription activator like effectors (TALEs). TALEs consist of 34 aa repeats (SEQ ID NO:1) at the core of their sequence. Each repeat corresponds to a base in the target DNA that is bound by the TALE, with one example shown as SEQ ID NO:2. Repeats differ only by 2 variable amino acids at positions 12 and 13. The code of this correspondence has been elucidated (Boch, J et al., Science, 2009 and Moscou, M et al., Science, 2009) and is shown in this figure. Applicants have developed a method for the synthesis of designer TALEs incorporating this code and capable of binding a sequence of choice within the genome (Zhang, F et al., Nature Biotechnology, 2011).

FIG. 3 shows a design of a LITE: TALE/Cryptochrome transcriptional activation. Each LITE is a two-component system which may comprise a TALE fused to CRY2 and the cryptochrome binding partner CIB1 fused to VP64, a transcription activator. In the inactive state, the TALE localizes its fused CRY2 domain to the promoter region of the gene of interest. At this point, CIB1 is unable to bind CRY2, leaving the CIB1-VP64 unbound in the nuclear space. Upon stimulation with 488 nm (blue) light, CRY2 undergoes a conformational change, revealing its CIB1 binding site (Liu, H et al., Science, 2008). Rapid binding of CIB1 results in recruitment of the fused VP64 domain, which induces transcription of the target gene.

FIG. 4 shows effects of cryptochrome dimer truncations on LITE activity. Truncations known to alter the activity of CRY2 and CIB1 (Kennedy M et al., Nature Methods 2010) were compared against the full length proteins. A LITE targeted to the promoter of Neurog2 was tested in Neuro-2a cells for each combination of domains. Following stimulation with 488 nm light, transcript levels of Neurog2 were quantified using qPCR for stimulated and unstimulated samples.

FIG. 5 shows a light-intensity dependent response of KLF4 LITE.

FIG. 6 shows activation kinetics of Neurog2 LITE and inactivation kinetics of Neurog2 LITE.

FIG. 7A shows the base-preference of various RVDs as determined using the Applicants' RVD screening system.

FIG. 7B shows the base-preference of additional RVDs as determined using the Applicants' RVD screening system.

FIGS. 8A-D show in (a) Natural structure of TALEs derived from Xanthomonas sp. Each DNA-binding module consists of 34 amino acids (SEQ ID NO:1), where the RVDs in the 12th and 13th amino acid positions of each repeat specify the DNA base being targeted (e.g., SEQ ID NO:2) according to the cipher NG=T, HD=C, NI=A, and NN=G or A. The DNA-binding modules are flanked by nonrepetitive N and C termini, which carry the translocation, nuclear localization (NLS) and transcription activation (AD) domains. A cryptic signal within the N terminus specifies a thymine as the first base of the target site. (b) The TALE toolbox allows rapid and inexpensive construction of custom TALE-TFs and TALENs. The kit consists of 12 plasmids in total: four monomer plasmids to be used as templates for PCR amplification, four TALE-TF and four TALEN cloning backbones corresponding to four different bases targeted by the 0.5 repeat. CMV, cytomegalovirus promoter; N term, nonrepetitive N terminus from the Hax3 TALE; C term, nonrepetitive C terminus from the Hax3 TALE; BsaI, type IIs restriction sites used for the insertion of custom TALE DNA-binding domains; ccdB+CmR, negative selection cassette containing the ccdB negative selection gene and chloramphenicol resistance gene; NLS, nuclear localization signal; VP64, synthetic transcriptional activator derived from VP16 protein of herpes simplex virus; 2A, 2A self-cleavage linker; EGFP, enhanced green fluorescent protein; polyA signal, polyadenylation signal; FokI, catalytic domain from the FokI endonuclease. (c) TALEs may be used to generate custom TALE-TFs and modulate the transcription of endogenous genes from the genome. The TALE DNA-binding domain is fused to the synthetic VP64 transcriptional activator, which recruits RNA polymerase and other factors needed to initiate transcription. (d) TALENs may be used to generate site-specific double-strand breaks to facilitate genome editing through nonhomologous repair or homology directed repair. Two TALENs target a pair of binding sites flanking a 16-bp spacer. The left and right TALENs recognize the top and bottom strands of the target sites, respectively. Each TALE DNA-binding domain is fused to the catalytic domain of FokI endonuclease; when FokI dimerizes, it cuts the DNA in the region between the left and right TALEN-binding sites.

FIG. 9A-F shows a table listing monomer sequences (excluding the RVDs at positions 12 and 13) (SEQ ID NOS:3-74) and the frequency with which monomers having a particular sequence occur.

FIG. 10 shows the comparison of the effect of non-RVD amino acid on TALE activity (SEQ ID NO:1 and variants thereof).

FIG. 11 shows an activator screen comparing levels of activation between VP64, p65 and VP16.

FIGS. 12A-D show the development of a TALE transcriptional repressor architecture. (a) Design of SOX2 TALE for TALE repressor screening. A TALE targeting a 14 bp sequence within the SOX2 locus of the human genome (SEQ ID NO:75) was synthesized. (b) List of all repressors screened and their host origin (left). Eight different candidate repressor domains were fused to the C-term of the SOX2 TALE. (c) The fold decrease of endogenous SOX2 mRNA is measured using qRTPCR by dividing the SOX2 mRNA levels in mock transfected cells by SOX2 mRNA levels in cells transfected with each candidate TALE repressor. (d) Transcriptional repression of endogenous CACNA1C. TALEs using NN, NK, and NH as the G-targeting RVD were constructed to target a 18 bp target site (SEQ ID NO:76) within the human CACNA1C locus. Each TALE is fused to the SID repression domain. NLS, nuclear localization signal; KRAB, Krüppel-associated box; SID, mSin interaction domain. All results are collected from three independent experiments in HEK 293FT cells. Error bars indicate s.e.m.; n=3. *p<0.05, Student's t test.

FIGS. 13A-C shows the optimization of TALE transcriptional repressor architecture using SID and SID4X. (a) Design of p11 TALE for testing of TALE repressor architecture. A TALE targeting a 20 bp sequence (p11 TALE binding site, SEQ ID NO:77) within the p11 (s100a10) locus of the mouse (Mus musculus) genome was synthesized. (b) Transcriptional repression of endogenous mouse p11 mRNA. TALEs targeting the mouse p11 locus harboring two different truncations of the wild type TALE architecture were fused to different repressor domains as indicated on the x-axis. The value in the bracket indicate the number of amino acids at the N- and C-termini of the TALE DNA binding domain flanking the DNA binding repeats, followed by the repressor domain used in the construct. The endogenous p11 mRNA levels were measured using qRT-PCR and normalized to the level in the negative control cells transfected with a GFP-encoding construct. (c) Fold of transcriptional repression of endogenous mouse p11. The fold decrease of endogenous p11 mRNA is measured using qRT-PCR through dividing the p11 mRNA levels in cells transfected with a negative control GFP construct by p11 mRNA levels in cells transfected with each candidate TALE repressors. The labeling of the constructs along the x-axis is the same as previous panel. NLS, nuclear localization signal; SID, mSin interaction domain; SID4X, an optimized four-time tandem repeats of SID domain linked by short peptide linkers. All results are collected from three independent experiments in Neuro2A cells. Error bars indicate s.e.m.; n=3. ***p<0.001, Student's t test.

FIG. 14A-D shows a comparison of two different types of TALE architecture.

FIGS. 15A-C show a chemically inducible TALE ABA inducible system. ABI (ABA insensitive 1) and PYL (PYL protein: pyrabactin resistance (PYR)/PYR1-like (PYL)) are domains from two proteins listed below that will dimerize upon binding of plant hormone Abscisic Acid (ABA). This plant hormone is a small molecule chemical that Applicants used in Applicants' inducible TALE system. In this system, the TALE DNA-binding polypeptide is fused to the ABI domain, whereas the VP64 activation domain or SID repressor domain or any effector domains are linked to the PYL domain. Thus, upon the induction by the presence of ABA molecule, the two interacting domains, ABI and PYL, will dimerize and allow the TALE to be linked to the effector domains to perform its activity in regulating target gene expression.

FIGS. 16A-B show a chemically inducible TALE 4OHT inducible system.

FIG. 17 depicts an effect of cryptochrome2 heterodimer orientation on LITE functionality.

FIG. 18 depicts mGlur2 LITE activity in mouse cortical neuron culture.

FIG. 19 depicts transduction of primary mouse neurons with LITE AAV vectors.

FIG. 20 depicts expression of LITE component in vivo.

FIG. 21 depicts an improved design of the construct where the specific NES peptide sequence used is LDLASLIL.

FIG. 22 depicts Sox2 mRNA levels in the absence and presence of 40H tamoxifen.

FIGS. 23A-E depict a Type II CRISPR locus from Streptococcus pyogenes SF370 can be reconstituted in mammalian cells to facilitate targeted DSBs of DNA. (A) Engineering of SpCas9 and SpRNase III with NLSs enables import into the mammalian nucleus. (B) Mammalian expression of SpCas9 and SpRNase III are driven by the EF1a promoter, whereas tracrRNA and pre-crRNA array (DR-Spacer-DR) are driven by the U6 promoter. A protospacer (blue highlight) from the human EMX1 locus (SEQ ID NO:78) with PAM is used as template for the spacer in the pre-crRNA array. (C) Schematic representation of base pairing between target locus (SEQ ID NOS:79-80) and EMX1-targeting crRNA (SEQ ID NO:81). Red arrow indicates putative cleavage site. (D) SURVEYOR assay for SpCas9-mediated indels. (E) An example chromatogram showing a micro-deletion, as well as representative sequences of mutated alleles (SEQ ID NOS:82-89) identified from 187 clonal amplicons. Red dashes, deleted bases; red bases, insertions or mutations. Scale bar=10 μm.

FIGS. 24A-C depict a SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. (A) Schematic of the human EMX1 locus (SEQ ID NOS:90-91) showing the location of five protospacers, indicated by blue lines with corresponding PAM in magenta. (B) Schematic of the pre-crRNA:tracrRNA complex (SEQ ID NOS:92-93) (top) showing hybridization between the direct repeat (gray) region of the pre-crRNA and tracrRNA. Schematic of a chimeric RNA design (SEQ ID NO:94) (M. Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816 (Aug. 17, 2012)) (bottom). tracrRNA sequence is shown in red and the 20 bp spacer sequence in blue. (C) SURVEYOR assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus. Each protospacer is targeted using either processed pre-crRNA:tracrRNA complex (crRNA) or chimeric RNA (chiRNA).

FIGS. 25A-D depict an evaluation of the SpCas9 specificity and comparison of efficiency with TALENs. (A) EMX1-targeting chimeric crRNAs with single point mutations were generated to evaluate the effects of spacer-protospacer mismatches (SEQ ID NOS:95-96, 97-108). (B) SURVEYOR assay comparing the cleavage efficiency of different mutant chimeric RNAs. (C) Schematic showing the design of TALENs targeting EMX1 (SEQ ID NOS:95-96). (D) SURVEYOR gel comparing the efficiency of TALEN and SpCas9 (N=3).

FIGS. 26A-G depict applications of Cas9 for homologous recombination and multiplex genome engineering. (A) Mutation of the RuvC I domain converts Cas9 into a nicking enzyme (SpCas9n) (B) Co-expression of EMX1-targeting chimeric RNA with SpCas9 leads to indels, whereas SpCas9n does not (N=3). (C) Schematic representation of the recombination strategy. A repair template is designed to insert restriction sites into EMX1 locus. Primers used to amplify the modified region are shown as red arrows. (D) Restriction fragments length polymorphism gel analysis. Arrows indicate fragments generated by HindIII digestion. (E) Example chromatogram showing successful recombination (SEQ ID NO:109). (F) SpCas9 can facilitate multiplex genome modification using a crRNA array containing two spacers (SEQ ID NOS:110, 111) targeting EMX1 and PVALB. Schematic showing the design of the crRNA array (top). Both spacers mediate efficient protospacer cleavage (bottom). (G) SpCas9 can be used to achieve precise genomic deletion. Two spacers (SEQ ID NOS:112, 113) targeting EMX1 (top) mediated a 118 bp genomic deletion (SEQ ID NOS:114-118) (bottom).

FIG. 27 depicts a schematic of the type II CRISPR-mediated DNA double-strand break. The type II CRISPR locus from Streptococcus pyogenes SF370 contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of nonrepetitive sequences (spacers, 30 bp each) (15-18, 30, 31). Each spacer is typically derived from foreign genetic material (protospacer), and directs the specificity of CRISPR-mediated nucleic acid cleavage. In the target nucleic acid, each protospacer is associated with a protospacer adjacent motif (PAM) whose recognition is specific to individual CRISPR systems (22, 23). The Type II CRISPR system carries out targeted DNA double-strand break (DSB) in sequential steps (M. Jinek et al., Science 337, 816 (Aug. 17, 2012); Gasiunas, R. et al. Proc Natl Acad Sci USA 109, E2579 (Sep. 25, 2012); J. E. Garneau et al., Nature 468, 67 (Nov. 4, 2010); R. Sapranauskas et al., Nucleic Acids Res 39, 9275 (November, 2011); A. H. Magadan et al. PLoS One 7, e40913 (2012)). First, the pre-crRNA array and tracrRNA are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA and associates with Cas9 as a duplex, which mediates the processing of the pre-crRNA into mature crRNAs containing individual, truncated spacer sequences. Third, the mature crRNA:tracrRNA duplex directs Cas9 to the DNA target consisting of the protospacer and the requisite PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer.

FIGS. 28A-C depict a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting. (A) Schematic showing the design and sequences of two tracrRNA transcripts (SEQ ID NOS:119-120) tested (short and long). Each transcript is driven by a U6 promoter. Transcription start site is marked as +1 and transcription terminator is as indicated. Blue line indicates the region whose reverse-complement sequence is used to generate northern blot probes for tracrRNA detection. (B) SURVEYOR assay comparing the efficiency of hSpCas9-mediated cleavage of the EMX1 locus. Two biological replicas are shown for each tracrRNA transcript. (C) Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying long or short tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III respectively. U6 indicate loading control blotted with a probe targeting human U6 snRNA. Transfection of the short tracrRNA expression construct led to abundant levels of the processed form of tracrRNA (˜75 bp) (E. Deltcheva et al., Nature 471, 602 (Mar. 31, 2011)). Very low amounts of long tracrRNA are detected on the Northern blot. As a result of these experiments, Applicants chose to use short tracrRNA for application in mammalian cells.

FIG. 29 depicts a SURVEYOR assay for detection of double strand break-induced micro insertions and deletions (D. Y. Guschin et al. Methods Mol Biol 649, 247 (2010)). Schematic of the SURVEYOR assay used to determine Cas9-mediated cleavage efficiency. First, genomic PCR (gPCR) is used to amplify the Cas9 target region from a heterogeneous population of modified and unmodified cells, and the gPCR products are reannealed slowly to generate heteroduplexes. The reannealed heteroduplexes are cleaved by SURVEYOR nuclease, whereas homoduplexes are left intact. Cas9-mediated cleavage efficiency (% indel) is calculated based on the fraction of cleaved DNA.

FIG. 30A-B depict a Northern blot analysis of crRNA processing in mammalian cells. (A) Schematic showing the expression vector for a single spacer flanked by two direct repeats (DR-EMX1(1)-DR) (SEQ ID NO: 121). The 30 bp spacer targeting the human EMX1 locus protospacer 1 (Table 1) is shown in blue and direct repeats are in shown in gray. Orange line indicates the region whose reverse complement sequence is used to generate northern blot probes for EMX1(1) crRNA detection. (B) Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III respectively. DR-EMX1(1)-DR was processed into mature crRNAs only in the presence of SpCas9 and short tracrRNA, and was not dependent on the presence of SpRNase III. The mature crRNA detected from transfected 293FT total RNA is ˜33 bp and is shorter than the 39-42 bp mature crRNA from S. pyogenes (E. Deltcheva et al., Nature 471, 602 (Mar. 31, 2011)), suggesting that the processed mature crRNA in human 293FT cells is likely different from the bacterial mature crRNA in S. pyogenes.

FIG. 31A-B depict bicistronic expression vectors for pre-crRNA array or chimeric crRNA with Cas9 (SEQ ID NOS:122-129). (A) Schematic showing the design of an expression vector for the pre-crRNA array. Spacers can be inserted between two BbsI sites using annealed oligonucleotides. Sequence design for the oligonucleotides are shown below with the appropriate ligation adapters indicated. (B) Schematic of the expression vector for chimeric crRNA. The guide sequence can be inserted between two BbsI sites using annealed oligonucleotides. The vector already contains the partial direct repeat (gray) and partial tracrRNA (red) sequences. WPRE, Woodchuck hepatitis virus posttranscriptional regulatory element.

FIGS. 32A-B depict a selection of protospacers in the human PVALB (SEQ ID NOS: 130-131) and mouse (SEQ ID NOS: 132-133) Th loci. Schematic of the human PVALB (A) and mouse Th (B) loci and the location of the three protospacers within the last exon of the PVALB and Th genes, respectively. The 30 bp protospacers are indicated by black lines and the adjacent PAM sequences are indicated by the magenta bar. Protospacers on the sense and anti-sense strands are indicated above and below the DNA sequences respectively.

FIGS. 33A-C depict occurrences of PAM sequences in the human genome. Histograms of distances between adjacent Streptococcus pyogenes SF370 locus 1 PAM (NGG) (A) and Streptococcus thermophilus LMD9 locus 1 PAM (NNAGAAW) (B) in the human genome. (C) Distances for each PAM by chromosome. Chr, chromosome. Putative targets were identified using both the plus and minus strands of human chromosomal sequences. Given that there may be chromatin, DNA methylation-, RNA structure, and other factors that may limit the cleavage activity at some protospacer targets, it is important to note that the actual targeting ability might be less than the result of this computational analysis.

FIGS. 34A-D depict type II CRISPR from Streptococcus thermophilus LMD-9 can also function in eukaryotic cells. (A) Schematic of CRISPR locus 2 from Streptococcus thermophilus LMD-9. (B) Design of the expression system for the S. thermophilus CRISPR system. Human codon-optimized hStCas9 is expressed using a constitutive EF1a promoter. Mature versions of tracrRNA and crRNA are expressed using the U6 promoter to ensure precise transcription initiation. Sequences for the mature crRNA and tracrRNA are shown (SEQ ID NOS: 134-135). A single based indicated by the lower case “a” in the crRNA sequence was used to remove the polyU sequence, which serves as a RNA Pol III transcriptional terminator. (C) Schematic showing protospacer and corresponding PAM sequences targets in the human EMX1 locus (SEQ ID NOS: 136-137). Two protospacer sequences are highlighted and their corresponding PAM sequences satisfying the NNAGAAW motif (SEQ ID NO:138) are indicated by magenta lines. Both protospacers are targeting the anti-sense strand. (D) SURVEYOR assay showing StCas9-mediated cleavage in the target locus. RNA guide spacers 1 and 2 induced 14% and 6.4% respectively. Statistical analysis of cleavage activity across biological replica at these two protospacer sites can be found in Table 1.

FIGS. 35A-E depict design and optimization of the LITE system. (A) A TALE DNA-binding domain (SEQ ID NO: 139) is fused to CRY2 and a transcriptional effector domain is fused to CIB1. In the inactive state, TALE-CRY2 binds the promoter region of the target gene while CIB1-effector remains unbound in the nucleus. The VP64 transcriptional activator is shown above. Upon illumination with blue light, TALE-CRY2 and CIB1-effector rapidly dimerize, recruiting CIB1-effector to the target promoter. The effector in turn modulates transcription of the target gene. (B) Light-dependent upregulation of the endogenous target Ngn2 mRNA with LITEs containing functional truncations of its light-sensitive binding partners. LITE-transfected Neuro-2a cells were stimulated for 24 h with 466 nm light at an intensity of 5 mW/cm² and a duty cycle of 7% (1 s pulses at 0.066 Hz). (C) Ngn2 upregulation with and without light by LITEs using different transcriptional activation domains VP16, VP64, and p65. Stimulation parameters are the same as (b). (D) The transcriptional activity of CRY2PHR-CIB1 LITE was found to vary according to the intensity of 466 nm blue light. Neuro 2a cells were stimulated for 24 h hours at a 7% duty cycle (1 s pulses at 0.066 Hz) (E) Light-induced toxicity measured as the percentage of cells positive for red-fluorescent ethidium homodimer-1 versus calcein-positive cells. All Ngn2 mRNA levels were measured relative to cells expressing YFP only (mean±s.e.m.; n=3-4)

FIGS. 36A-B depict kinetics of light-induced transcriptional activation. (A) Time course of light-dependent Ngn2 upregulation by TALE-CRY2PHR and CIB1-VP64 LITEs. LITE-transfected Neuro-2a cells were stimulated with 466 nm light at an intensity of 5 mW/cm² and a duty cycle of 7% (1 s pulses at 0.066 Hz). (B) Decrease of Ngn2 mRNA levels after 6 h of light stimulation. All Ngn2 mRNA levels were measured relative to expressing YFP control cells (mean±s.e.m.; n=3-4) (*=p<0.05 and ***=p<0.001).

FIGS. 37A-F depict virus-mediated TALE delivery enabling bimodal control of endogenous gene expression in neurons (A) General schematic of constitutive TALE transcriptional activator and repressor packaged into AAV. Effector domains VP64 and SID4X are highlighted. (B) Representative images showing transduction with AAV-TALE-VP64 constructs from (a) in primary cortical neurons. Cells were stained for virally delivered GFP and neuronal marker NeuN. Scale bars=25 μm. (C) 6 TALEs were designed, with two TALEs targeting each of the endogenous mouse loci Grm5, Grin2a, and Grm2 (SEQ ID NOS:140-145). TALEs were fused to the transcriptional activator domain VP64 or the repressor domain SID4X and virally transduced into primary neurons. Both the target gene upregulation via VP64 and downregulation via SID4X are shown for each TALE relative to levels in neurons expressing GFP only. (D) Efficient delivery of TALE-VP64 by AAV into the ILC of mice. Scale bar=100 um. (Cg1=cingulate cortex, PLC=prelimbic cortex, ILC=infralimbic cortex). (E) Higher magnification image of efficient transduction of neurons in ILC. (F) Grm2 mRNA upregulation by TALE-VP64 in vivo in ILC (mean±s.e.m.; n=3).

FIGS. 38A-J depict light-mediated manipulation of Grm2 expression in primary neurons and in vivo (A) AAV LITE activator construct with switched CRY2PHR and CIB1 architecture. (B) Representative images showing co-transduction of AAV-delivered LITE constructs in primary neurons. Cells were stained for GFP, HA-tag, and DAPI. (Scale bars=25 μm). (C) Light-induced activation of Grm2 expression in primary neurons after 24 h of stimulation with 0.8% duty cycle pulsed 466 nm light (250 ms pulses at 0.033 Hz or 500 ms pulses at 0.016 Hz; 5 mW/cm²). (D) Upregulation of Grm2 mRNA in primary cortical neurons with and without light stimulation at 4 h and 24 h time points. Expression levels are shown relative to neurons transduced with GFP only. (E) Quantification of mGluR2 protein levels in GFP only control transductions, unstimulated neurons with LITEs, and light-stimulated neurons with LITEs. A representative western blot is shown with β-tubulin-III as a loading control. (F) LITE repressor construct highlighting SID4X repressor domain. (G) Light-induced repression of endogenous Grm2 expression in primary cortical neurons using Grm2 T1-LITE and Grm2 T2-LITE. Fold downregulation is shown relative to neurons transduced with GFP only (mean±s.e.m.; n=3-4 for all subpanels). (H) Schematic showing transduction of ILC with the LITE system, the optical fiber implant, and the 0.35 mm diameter brain punch used for tissue isolation. (I) Representative images of ILC co-transduced with both LITE components. Stains are shown for HA-tag (red), GFP (green), and DAPI (blue). (Scale bar=25 μm). (J) Light-induced activation of endogenous Grm2 expression using LITEs transduced into ILC.

FIG. 39 depicts an activation Ratio of CRY2 and CIB1 truncations. Fold activation of Ngn2 expression by LITEs was calculated as the ratio of mRNA levels in stimulated cells versus unstimulated cells (light/no light; experiment and data corresponding to FIG. 35B), for each CRY2 and CIB1 truncation pair.

FIG. 40 depicts an impact of illumination duty cycle on LITE-mediated gene expression. Varying duty cycles (illumination as percentage of total time) were used to stimulate HEK293FT cells expressing LITEs targeting the KLF4 gene, in order to investigate the effect of duty cycle on LITE activity. KLF4 expression levels were compared to cells expressing GFP only. Stimulation parameters were: 466 nm, 5 mW/cm² for 24 h. Pulses were performed at 0.067 Hz with the following durations: 1.7%=0.25 s pulse, 7%=1 s pulse, 27%=4 s pulse, 100%=constant illumination.

FIG. 41 depicts an illustration of the absorption spectrum of CRY2 in vitro. Cryptochrome 2 was optimally activated by 350-475 nm light′. A sharp drop in absorption and activation was seen for wavelengths greater than 480 nm. Spectrum was adapted from Banerjee, R. et al. The Signaling State of Arabidopsis Cryptochrome 2 Contains Flavin Semiquinone. Journal of Biological Chemistry 282, 14916-14922, doi:10.1074/jbc.M700616200 (2007).

FIGS. 42A-C depict AAV supernatant production. (A) Lentiviral and AAV vectors carrying GFP were used to test transduction efficiency. (B) Primary embryonic cortical neurons were transduced with 250 μL supernatant derived from the same number of AAV or lentivirus-transfected 293FT cells. Representative images of GFP expression were collected at 7 d.p.i. Scale bars=50 μm. (C) The depicted process was developed for the production of AAV supernatant and subsequent transduction of primary neurons. 293FT cells were transfected with an AAV vector carrying the gene of interest, the AAV1 serotype packaging vector (pAAV1), and helper plasmid (pDF6) using PEI. 48 h later, the supernatant was harvested and filtered through a 0.45 μm PVDF membrane. Primary neurons were then transduced with supernatant and remaining aliquots were stored at −80° C. Stable levels of AAV construct expression were reached after 5-6 days.

FIG. 43 depicts a selection of TALE target sites guided by DNaseI-sensitive chromatin regions. High DNaseI sensitivity based on mouse cortical tissue data from ENCODE (at the website of genome.ucsc.edu) was used to identify open chromatin regions. The peak with the highest amplitude within the region 2 kb upstream of the transcriptional start site was selected for targeting. TALE binding targets were then picked within a 200 bp region at the center of the peak.

FIG. 44 depicts a TALE SID4X repressor characterization. A synthetic repressor was constructed by concatenating 4 SID domains (SID4X). To identify the optimal TALE-repressor architecture, SID or SID4X was fused to a TALE designed to target the mouse p11 gene (SEQ ID NO:146). Fold decrease in p11 mRNA was assayed using qRT-PCR.

FIGS. 45A-B depict exchanging CRY2PHR and CIB1 components. (A) TALE-CIB1::CRY2PHR-VP64 was able to activate Ngn2 at higher levels than TALE-CRY2PHR::CIB1-VP64. (B) Fold activation ratios (light versus no light) ratios of Ngn2 LITEs show similar efficiency for both designs. Stimulation parameters were the same as those used in FIG. 35B.

FIG. 46 depicts an impact of light duty cycle on primary neuron health. The effect of light stimulation on primary cortical neuron health was compared for duty cycles of 7%, 0.8%, and no light conditions. Calcein was used to evaluate neuron viability. Bright-field images were captured to show morphology and cell integrity. Primary cortical neurons were stimulated with the indicated duty cycle for 24 h with 5 mW/cm² of 466 nm light. Representative images, scale bar=50 μm. Pulses were performed in the following manner: 7% duty cycle=1 s pulse at 0.067 Hz, 0.8% duty cycle=0.5 s pulse at 0.0167 Hz.

FIGS. 47A-B depict a contribution of individual LITE components to baseline transcription modulation. (A) Grm2 mRNA levels were determined in primary neurons transfected with individual LITE components. Primary neurons expressing T6-CIB1 alone led to a similar increase in Grm2 mRNA levels as unstimulated cells expressing the complete LITE system. (B) Transcription repression by individual LITE repressor components targeting the Grm2 gene was compared.

FIG. 48 depicts a co-transduction efficiency of LITE components by AAV1/2 in mouse infralimbic cortex. Cells transduced by T6-CIB1 alone, CRY2PHR-VP64 alone, or co-transduced were calculated as a percentage of all transduced cells.

FIG. 49 shows a schematic of an AAV-promotor-TALE-effector construct. In the construct: hSyn=human synapsin 1 promoter; N+136=TALE N-term, AA+136 truncation; C63=TALE C-term, AA+63 truncation; vp=VP64 effector domain; GFP=green fluorescent protein; WPRE=Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element; bGH=bovine growth hormone polyA; ITR=AAV inverted terminal repeat; AmpR=ampicillin resistance gene.

DETAILED DESCRIPTION OF THE INVENTION

The term “nucleic acid” or “nucleic acid sequence” refers to a deoxyribonucleic or ribonucleic oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. The term also encompasses nucleic-acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996.

As used herein, “recombinant” refers to a non-naturally occurring composition comprising materials from more than one origin and, in some embodiments, materials derived from more than one organism. A “recombinant construct” may be a polynucleotide synthesized or otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), and the invention includes methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. “Recombinant means” encompasses methods of recombining compositions, e.g., ligation of nucleic acids having various coding regions or domains or promoter sequences from different sources into an expression cassette or vector for expression of, e.g., inducible or constitutive expression of polypeptide coding sequences in the vectors of invention.

The term “heterologous” when used with reference to a nucleic acid, indicates that the nucleic acid is in a cell or a virus where it is not normally found in nature; or, comprises two or more subsequences that are not found in the same relationship to each other as normally found in nature, or is recombinantly engineered so that its level of expression, or physical relationship to other nucleic acids or other molecules in a cell, or structure, is not normally found in nature. A similar term used in this context is “exogenous”. For instance, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged in a manner not found in nature; e.g., a human gene operably linked to a promoter sequence inserted into an adenovirus-based vector of the invention. As an example, a heterologous nucleic acid of interest may encode an immunogenic gene product, wherein the adenovirus is administered therapeutically or prophylactically as a carrier or drug-vaccine composition. Heterologous sequences may comprise various combinations of promoters and sequences, examples of which are described in detail herein.

A “therapeutic ligand” may be a substance which may bind to a receptor of a target cell with therapeutic effects.

A “therapeutic effect” may be a consequence of a medical treatment of any kind, the results of which are judged by one of skill in the field to be desirable and beneficial. The “therapeutic effect” may be a behavioral or physiologic change which occurs as a response to the medical treatment. The result may be expected, unexpected, or even an unintended consequence of the medical treatment. A “therapeutic effect” may include, for example, a reduction of symptoms in a subject suffering from infection by a pathogen.

A “target cell” may be a cell in which an alteration in its activity may induce a desired result or response.

A “ligand” may be any substance that binds to and forms a complex with a biomolecule to serve a biological purpose. As used herein, “ligand” may also refer to an “antigen” or “immunogen”. As used herein “antigen” and “immunogen” are used interchangeably.

“Expression” of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context.

As used herein, a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. By way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The present invention comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinant constructs thereof.

With respect to exogenous DNA for expression in a vector (e.g., encoding an epitope of interest and/or an antigen and/or a therapeutic) and documents providing such exogenous DNA, as well as with respect to the expression of transcription and/or translation factors for enhancing expression of nucleic acid molecules, and as to terms such as “epitope of interest”, “therapeutic”, “immune response”, “immunological response”, “protective immune response”, “immunological composition”, “immunogenic composition”, and “vaccine composition”, inter alia, reference is made to U.S. Pat. No. 5,990,091 issued Nov. 23, 1999, and WO 98/00166 and WO 99/60164, and the documents cited therein and the documents of record in the prosecution of that patent and those PCT applications; all of which are incorporated herein by reference. Thus, U.S. Pat. No. 5,990,091 and WO 98/00166 and WO 99/60164 and documents cited therein and documents of record in the prosecution of that patent and those PCT applications, and other documents cited herein or otherwise incorporated herein by reference, may be consulted in the practice of this invention; and, all exogenous nucleic acid molecules, promoters, and vectors cited therein may be used in the practice of this invention. In this regard, mention is also made of U.S. Pat. Nos. 6,706,693; 6,716,823; 6,348,450; U.S. patent application Ser. Nos. 10/424,409; 10/052,323; 10/116,963; 10/346,021; and WO 99/08713, published Feb. 25, 1999, from PCT/US98/16739.

As used herein, the terms “drug composition” and “drug”, “vaccinal composition”, “vaccine”, “vaccine composition”, “therapeutic composition” and “therapeutic-immunologic composition” cover any composition that induces protection against an antigen or pathogen. In some embodiments, the protection may be due to an inhibition or prevention of infection by a pathogen. In other embodiments, the protection may be induced by an immune response against the antigen(s) of interest, or which efficaciously protects against the antigen; for instance, after administration or injection into the subject, elicits a protective immune response against the targeted antigen or immunogen or provides efficacious protection against the antigen or immunogen expressed from the inventive adenovirus vectors of the invention. The term “pharmaceutical composition” means any composition that is delivered to a subject. In some embodiments, the composition may be delivered to inhibit or prevent infection by a pathogen.

A “therapeutically effective amount” is an amount or concentration of the recombinant vector encoding the gene of interest, that, when administered to a subject, produces a therapeutic response or an immune response to the gene product of interest.

The term “viral vector” as used herein includes but is not limited to retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and herpes simplex virus.

The present invention enables spatiotemporal control of endogenous gene expression using a form of energy. The form of energy by include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy. In a preferred embodiment of the invention, the form of energy is electromagnetic radiation, preferably, light energy. Previous approaches to control expression of endogenous genes, such as transcription activators linked to DNA binding zinc finger proteins provided no mechanism for temporal or spatial control. The capacity for photoactivation of the system described herein allows the induction of gene expression modulation to begin at a precise time within a localized population of cells.

Two key molecular tools were leveraged in the design of the photoresponsive transcription activator-like (TAL) effector system. First, the DNA binding specificity of engineered TAL effectors is utilized to localize the complex to a particular region in the genome. Second, light-induced protein dimerization is used to attract an activating or repressing domain to the region specified by the TAL effector, resulting in modulation of the downstream gene.

Inducible effectors are contemplated for in vitro or in vivo application in which temporally or spatially specific gene expression control is desired. In vitro examples: temporally precise induction/suppression of developmental genes to elucidate the timing of developmental cues, spatially controlled induction of cell fate reprogramming factors for the generation of cell-type patterned tissues. In vivo examples: combined temporal and spatial control of gene expression within specific brain regions.

In a preferred embodiment of the invention, the inducible effector is a Light Inducible Transcriptional Effector (LITE). The modularity of the LITE system allows for any number of effector domains to be employed for transcriptional modulation. In a particularly advantageous embodiment, transcription activator like effector (TALE) and the activation domain VP64 are utilized in the present invention.

LITEs are designed to modulate or alter expression of individual endogenous genes in a temporally and spatially precise manner. Each LITE may comprise a two component system consisting of a customized DNA-binding transcription activator like effector (TALE) protein, a light-responsive cryptochrome heterodimer from Arabadopsis thaliana, and a transcriptional activation/repression domain. The TALE is designed to bind to the promoter sequence of the gene of interest. The TALE protein is fused to one half of the cryptochrome heterodimer (cryptochrome-2 or CIB1), while the remaining cryptochrome partner is fused to a transcriptional effector domain. Effector domains may be either activators, such as VP16, VP64, or p65, or repressors, such as KRAB, EnR, or SID. In a LITE's unstimulated state, the TALE-cryptochrome2 protein localizes to the promoter of the gene of interest, but is not bound to the CIB1-effector protein. Upon stimulation of a LITE with blue spectrum light, cryptochrome-2 becomes activated, undergoes a conformational change, and reveals its binding domain. CIB1, in turn, binds to cryptochrome-2 resulting in localization of the effector domain to the promoter region of the gene of interest and initiating gene overexpression or silencing.

Activator and repressor domains may selected on the basis of species, strength, mechanism, duration, size, or any number of other parameters. Preferred effector domains include, but are not limited to, a transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylases domain, nuclease domain, repressor domain, activator domain, nuclear-localization signal domains, transcription-protein recruiting domain, cellular uptake activity associated domain, nucleic acid binding domain or antibody presentation domain.

Gene targeting in a LITE or in any other inducible effector may be achieved via the specificity of customized TALE DNA binding proteins. A target sequence in the promoter region of the gene of interest is selected and a TALE customized to this sequence is designed. The central portion of the TALE consists of tandem repeats 34 amino acids in length. Although the sequences of these repeats are nearly identical, the 12th and 13th amino acids (termed repeat variable diresidues) of each repeat vary, determining the nucleotide-binding specificity of each repeat. Thus, by synthesizing a construct with the appropriate ordering of TALE monomer repeats, a DNA binding protein specific to the target promoter sequence is created.

In advantageous embodiments of the invention, the methods provided herein use isolated, non-naturally occurring, recombinant or engineered DNA binding proteins that comprise TALE monomers or TALE monomers or half monomers as a part of their organizational structure that enable the targeting of nucleic acid sequences with improved efficiency and expanded specificity.

Naturally occurring TALEs or “wild type TALEs” are nucleic acid binding proteins secreted by numerous species of proteobacteria. TALE polypeptides contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides that are predominantly 33, 34 or 35 amino acids in length and that differ from each other mainly in amino acid positions 12 and 13. In advantageous embodiments the nucleic acid is DNA. As used herein, the term “polypeptide monomers”, “TALE monomers” or “monomers” will be used to refer to the highly conserved repetitive polypeptide sequences within the TALE nucleic acid binding domain and the term “repeat variable di-residues” or “RVD” will be used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomers. As provided throughout the disclosure, the amino acid residues of the RVD are depicted using the IUPAC single letter code for amino acids. A general representation of a TALE monomer which is comprised within the DNA binding domain is X1-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid. X12X13 indicate the RVDs. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent and in such monomers, the RVD consists of a single amino acid. In such cases the RVD may be alternatively represented as X*, where X represents X12 and (*) indicates that X13 is absent. The DNA binding domain comprises several repeats of TALE monomers and this may be represented as (X1-11-(X12X13)-X14-33 or 34 or 35)z, where in an advantageous embodiment, z is at least 5 to 40. In a further advantageous embodiment, z is at least 10 to 26.

The TALE monomers have a nucleotide binding affinity that is determined by the identity of the amino acids in its RVD. For example, polypeptide monomers with an RVD of NI preferentially bind to adenine (A), monomers with an RVD of NG preferentially bind to thymine (T), monomers with an RVD of HD preferentially bind to cytosine (C) and monomers with an RVD of NN preferentially bind to both adenine (A) and guanine (G). In yet another embodiment of the invention, monomers with an RVD of IG preferentially bind to T. Thus, the number and order of the polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity. In still further embodiments of the invention, monomers with an RVD of NS recognize all four base pairs and may bind to A, T, G or C. The structure and function of TALEs is further described in, for example, Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated by reference in its entirety.

The polypeptides used in methods of the invention are isolated, non-naturally occurring, recombinant or engineered nucleic acid-binding proteins that have nucleic acid or DNA binding regions containing polypeptide monomer repeats that are designed to target specific nucleic acid sequences.

As described herein, polypeptide monomers having an RVD of HN or NH preferentially bind to guanine and thereby allow the generation of TALE polypeptides with high binding specificity for guanine containing target nucleic acid sequences. In a preferred embodiment of the invention, polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS preferentially bind to guanine. In a much more advantageous embodiment of the invention, polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH, SS and SN preferentially bind to guanine and thereby allow the generation of TALE polypeptides with high binding specificity for guanine containing target nucleic acid sequences. In an even more advantageous embodiment of the invention, polypeptide monomers having RVDs HH, KH, NH, NK, NQ, RH, RN and SS preferentially bind to guanine and thereby allow the generation of TALE polypeptides with high binding specificity for guanine containing target nucleic acid sequences. In a further advantageous embodiment, the RVDs that have high binding specificity for guanine are RN, NH RH and KH. Furthermore, polypeptide monomers having an RVD of NV preferentially bind to adenine and guanine. In more preferred embodiments of the invention, monomers having RVDs of H*, HA, KA, N*, NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and thymine with comparable affinity.

The predetermined N-terminal to C-terminal order of the one or more polypeptide monomers of the nucleic acid or DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the polypeptides of the invention will bind. As used herein the monomers and at least one or more half monomers are “specifically ordered to target” the genomic locus or gene of interest. In plant genomes, the natural TALE-binding sites always begin with a thymine (T), which may be specified by a cryptic signal within the nonrepetitive N-terminus of the TALE polypeptide; in some cases this region may be referred to as repeat 0. In animal genomes, TALE binding sites do not necessarily have to begin with a thymine (T) and polypeptides of the invention may target DNA sequences that begin with T, A, G or C. The tandem repeat of TALE monomers always ends with a half-length repeat or a stretch of sequence that may share identity with only the first 20 amino acids of a repetitive full length TALE monomer and this half repeat may be referred to as a half-monomer (FIG. 8). Therefore, it follows that the length of the nucleic acid or DNA being targeted is equal to the number of full monomers plus two.

As described in Zhang et al., Nature Biotechnology 29:149-153 (2011), TALE polypeptide binding efficiency may be increased by including amino acid sequences from the “capping regions” that are directly N-terminal or C-terminal of the DNA binding region of naturally occurring TALEs into the engineered TALEs at positions N-terminal or C-terminal of the engineered TALE DNA binding region. Thus, in certain embodiments, the TALE polypeptides described herein further comprise an N-terminal capping region and/or a C-terminal capping region.

An exemplary amino acid sequence of a N-terminal capping region is:

(SEQ ID NO: 147) M D P I R S R T P S P A R E L L S G P Q P D G V Q P T A D R G V S P P A G G P L D G L P A R R T M S R T R L P S P P A P S P A F S A D S F S D L L R Q F D P S L F N T S L F D S L P P F G A H H T E A A T G E W D E V Q S G L R A A D A P P P T M R V A V T A A R P P R A K P A P R R R A A Q P S D A S P A A Q V D L R T L G Y S Q Q Q Q E K I K P K V R S T V A Q H H E A L V G H G F T H A H I V A L S Q H P A A L G T V A V K Y Q D M I A A L P E A T H E A I V G V G K Q W S G A R A L E A L L T V A G E L R G P P L Q L D T G Q L L K I A K R G G V T A V E A V H A W R N A L T G A P L N

An exemplary amino acid sequence of a C-terminal capping region is:

(SEQ ID NO: 148) R P A L E S I V A Q L S R P D P A L A A L T N D H L V A L A C L G G R P A L D A V K K G L P H A P A L I K R T N R R I P E R T S H R V A D H A Q V V R V L G F F Q C H S H P A Q A F D D A M T Q F G M S R H G L L Q L F R R V G V T E L E A R S G T L P P A S Q R W D R I L Q A S G M K R A K P S P T S T Q T P D Q A S L H A F A D S L E R D L D A P S P M H E G D Q T R A S

As used herein the predetermined “N-terminus” to “C terminus” orientation of the N-terminal capping region, the DNA binding domain comprising the repeat TALE monomers and the C-terminal capping region provide structural basis for the organization of different domains in the d-TALEs or polypeptides of the invention.

The entire N-terminal and/or C-terminal capping regions are not necessary to enhance the binding activity of the DNA binding region. Therefore, in certain embodiments, fragments of the N-terminal and/or C-terminal capping regions are included in the TALE polypeptides described herein.

In certain embodiments, the TALE polypeptides described herein contain a N-terminal capping region fragment that included at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270 amino acids of an N-terminal capping region. In certain embodiments, the N-terminal capping region fragment amino acids are of the C-terminus (the DNA-binding region proximal end) of an N-terminal capping region. As described in Zhang et al., Nature Biotechnology 29:149-153 (2011), N-terminal capping region fragments that include the C-terminal 240 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 147 amino acids retain greater than 80% of the efficacy of the full length capping region, and fragments that include the C-terminal 117 amino acids retain greater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain a C-terminal capping region fragment that included at least 6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155, 160, 170, 180 amino acids of a C-terminal capping region. In certain embodiments, the C-terminal capping region fragment amino acids are of the N-terminus (the DNA-binding region proximal end) of a C-terminal capping region. As described in Zhang et al., Nature Biotechnology 29:149-153 (2011), C-terminal capping region fragments that include the C-terminal 68 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 20 amino acids retain greater than 50% of the efficacy of the full length capping region.

In certain embodiments, the capping regions of the TALE polypeptides described herein do not need to have identical sequences to the capping region sequences provided herein. Thus, in some embodiments, the capping region of the TALE polypeptides described herein have sequences that are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or share identity to the capping region amino acid sequences provided herein. Sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. In some preferred embodiments, the capping region of the TALE polypeptides described herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein.

Sequence homologies may be generated by any of a number of computer programs known in the art, which include but are not limited to BLAST or FASTA. Suitable computer program for carrying out alignments like the GCG Wisconsin Bestfit package may also be used. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

In advantageous embodiments described herein, the TALE polypeptides of the invention include a nucleic acid binding domain linked to the one or more effector domains. The terms “effector domain” or “regulatory and functional domain” refer to a polypeptide sequence that has an activity other than binding to the nucleic acid sequence recognized by the nucleic acid binding domain. By combining a nucleic acid binding domain with one or more effector domains, the polypeptides of the invention may be used to target the one or more functions or activities mediated by the effector domain to a particular target DNA sequence to which the nucleic acid binding domain specifically binds.

In some embodiments of the TALE polypeptides described herein, the activity mediated by the effector domain is a biological activity. For example, in some embodiments the effector domain is a transcriptional inhibitor (i.e., a repressor domain), such as an mSin interaction domain (SID). SID4X domain or a Krüppel-associated box (KRAB) or fragments of the KRAB domain. In some embodiments the effector domain is an enhancer of transcription (i.e. an activation domain), such as the VP16, VP64 or p65 activation domain. In some embodiments, the nucleic acid binding is linked, for example, with an effector domain that includes but is not limited to a transposase, integrase, recombinase, resolvase, invertase, protease, DNA methyltransferase, DNA demethylase, histone acetylase, histone deacetylase, nuclease, transcriptional repressor, transcriptional activator, transcription factor recruiting, protein nuclear-localization signal or cellular uptake signal.

In some embodiments, the effector domain is a protein domain which exhibits activities which include but are not limited to transposase activity, integrase activity, recombinase activity, resolvase activity, invertase activity, protease activity, DNA methyltransferase activity, DNA demethylase activity, histone acetylase activity, histone deacetylase activity, nuclease activity, nuclear-localization signaling activity, transcriptional repressor activity, transcriptional activator activity, transcription factor recruiting activity, or cellular uptake signaling activity. Other preferred embodiments of the invention may include any combination the activities described herein.

As described in Zhang et al., Nature Biotechnology 29:149-153 (2011), a TALE polypeptide having a nucleic acid binding domain and an effector domain may be used to target the effector domain's activity to a genomic position having a predetermined nucleic acid sequence recognized by the nucleic acid binding domain. In some embodiments of the invention described herein, TALE polypeptides are designed and used for targeting gene regulatory activity, such as transcriptional or translational modifier activity, to a regulatory, coding, and/or intergenic region, such as enhancer and/or repressor activity, that may affect transcription upstream and downstream of coding regions, and may be used to enhance or repress gene expression. For example, TALEs polypeptide may comprise effector domains having DNA-binding domains from transcription factors, effector domains from transcription factors (activators, repressors, co-activators, co-repressors), silencers, nuclear hormone receptors, and/or chromatin associated proteins and their modifiers (e.g., methylases, kinases, phosphatases, acetylases and deacetylases). In a preferred embodiment, the TALE polypeptide may comprise a nuclease domain. In a more preferred embodiment the nuclease domain is a non-specific FokI endonucleases catalytic domain.

In a further embodiment, useful domains for regulating gene expression may also be obtained from the gene products of oncogenes. In yet further advantageous embodiments of the invention, effector domains having integrase or transposase activity may be used to promote integration of exogenous nucleic acid sequence into specific nucleic acid sequence regions, eliminate (knock-out) specific endogenous nucleic acid sequence, and/or modify epigenetic signals and consequent gene regulation, such as by promoting DNA methyltransferase, DNA demethylase, histone acetylase and histone deacetylase activity. In other embodiments, effector domains having nuclease activity may be used to alter genome structure by nicking or digesting target sequences to which the polypeptides of the invention specifically bind, and may allow introduction of exogenous genes at those sites. In still further embodiments, effector domains having invertase activity may be used to alter genome structure by swapping the orientation of a DNA fragment.

In particularly advantageous embodiments, the polypeptides used in the methods of the invention may be used to target transcriptional activity. As used herein, the term “transcription factor” refers to a protein or polypeptide that binds specific DNA sequences associated with a genomic locus or gene of interest to control transcription. Transcription factors may promote (as an activator) or block (as a repressor) the recruitment of RNA polymerase to a gene of interest. Transcription factors may perform their function alone or as a part of a larger protein complex. Mechanisms of gene regulation used by transcription factors include but are not limited to a) stabilization or destabilization of RNA polymerase binding, b) acetylation or deacetylation of histone proteins and c) recruitment of co-activator or co-repressor proteins. Furthermore, transcription factors play roles in biological activities that include but are not limited to basal transcription, enhancement of transcription, development, response to intercellular signaling, response to environmental cues, cell-cycle control and pathogenesis. With regards to information on transcriptional factors, mention is made of Latchman and DS (1997) Int. J. Biochem. Cell Biol. 29 (12): 1305-12; Lee T I, Young R A (2000) Annu Rev. Genet. 34: 77-137 and Mitchell P J, Tjian R (1989) Science 245 (4916): 371-8, herein incorporated by reference in their entirety.

Light responsiveness of a LITE is achieved via the activation and binding of cryptochrome-2 and CIB1. As mentioned above, blue light stimulation induces an activating conformational change in cryptochrome-2, resulting in recruitment of its binding partner CIB1. This binding is fast and reversible, achieving saturation in <15 sec following pulsed stimulation and returning to baseline <15 min after the end of stimulation. These rapid binding kinetics result in a LITE system temporally bound only by the speed of transcription/translation and transcript/protein degradation, rather than uptake and clearance of inducing agents. Cryptochrome-2 activation is also highly sensitive, allowing for the use of low light intensity stimulation and mitigating the risks of phototoxicity. Further, in a context such as the intact mammalian brain, variable light intensity may be used to control the size of a LITE stimulated region, allowing for greater precision than vector delivery alone may offer.

The modularity of the LITE system allows for any number of effector domains to be employed for transcriptional modulation. Thus, activator and repressor domains may be selected on the basis of species, strength, mechanism, duration, size, or any number of other parameters.

Applicants next present two prototypical manifestations of the LITE system. The first example is a LITE designed to activate transcription of the mouse gene NEUROG2. The sequence TGAATGATGATAATACGA (SEQ ID NO:149), located in the upstream promoter region of mouse NEUROG2, was selected as the target and a TALE was designed and synthesized to match this sequence. The TALE sequence was linked to the sequence for cryptochrome-2 via a nuclear localization signal (amino acids: SPKKKRKVEAS; SEQ ID NO: 150) to facilitate transport of the protein from the cytosol to the nuclear space. A second vector was synthesized comprising the CIB1 domain linked to the transcriptional activator domain VP64 using the same nuclear localization signal. This second vector, also a GFP sequence, is separated from the CIB1-VP64 fusion sequence by a 2A translational skip signal. Expression of each construct was driven by a ubiquitous, constitutive promoter (CMV or EF1-α). Mouse neuroblastoma cells from the Neuro 2A cell line were co-transfected with the two vectors. After incubation to allow for vector expression, samples were stimulated by periodic pulsed blue light from an array of 488 nm LEDs. Unstimulated co-transfected samples and samples transfected only with the fluorescent reporter YFP were used as controls. At the end of each experiment, mRNA was purified from the samples analyzed via qPCR.

Truncated versions of cryptochrome-2 and CIB1 were cloned and tested in combination with the full-length versions of cryptochrome-2 and CIB1 in order to determine the effectiveness of each heterodimer pair. The combination of the CRY2PHR domain, consisting of the conserved photoresponsive region of the cryptochrome-2 protein, and the full-length version of CIB1 resulted in the highest upregulation of Neurog2 mRNA levels (˜22 fold over YFP samples and ˜7 fold over unstimulated co-transfected samples). The combination of full-length cryptochrome-2 (CRY2) with full-length CIB1 resulted in a lower absolute activation level (˜4.6 fold over YFP), but also a lower baseline activation (˜1.6 fold over YFP for unstimulated co-transfected samples). These cryptochrome protein pairings may be selected for particular uses depending on absolute level of induction required and the necessity to minimize baseline “leakiness” of the LITE system.

Speed of activation and reversibility are critical design parameters for the LITE system. The invention contemplates energy sources such as electromagnetic radiation, sound energy or thermal energy.

The cells of the present invention are preferably a eukaryotic cell, advantageously an animal cell, more advantageously a mammalian cell.

The present invention also contemplates a multiplex genome engineering using CRISPR/Cas systems. Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. Applicants engineered two different type II CRISPR systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Finally, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the CRISPR technology.

In general, “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, all or a portion of the tracr sequence may also form part of a CRISPR complex, such as by hybridization to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.

In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.

In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ (visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000″Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

In some embodiments, a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 151); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK; SEQ ID NO: 152); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 153) or RQRRNELKRSP (SEQ ID NO: 154); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 155); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 156) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 157) and PPKKARED (SEQ ID NO: 158) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 159) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 160) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 161) and PKQKKRK (SEQ ID NO: 162) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 163) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 164) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 165) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 166) of the steroid hormone receptors (human) glucocorticoid.

In general, the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.

The present invention also encompasses nucleic acid encoding the polypeptides of the present invention. The nucleic acid may comprise a promoter, advantageously human Synapsin I promoter (hSyn). In a particularly advantageous embodiment, the nucleic acid may be packaged into an adeno associated viral vector (AAV).

Also contemplated by the present invention are recombinant vectors and recombinant adenoviruses that may comprise subviral particles from more than one adenovirus serotype. For example, it is known that adenovirus vectors may display an altered tropism for specific tissues or cell types (Havenga, M. J. E. et al., 2002), and therefore, mixing and matching of different adenoviral capsids, i.e., fiber, or penton proteins from various adenoviral serotypes may be advantageous. Modification of the adenoviral capsids, including fiber and penton may result in an adenoviral vector with a tropism that is different from the unmodified adenovirus. Adenovirus vectors that are modified and optimized in their ability to infect target cells may allow for a significant reduction in the therapeutic or prophylactic dose, resulting in reduced local and disseminated toxicity.

Viral vector gene delivery systems are commonly used in gene transfer and gene therapy applications. Different viral vector systems have their own unique advantages and disadvantages. Viral vectors that may be used to express the pathogen-derived ligand of the present invention include but are not limited to adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, herpes simplex viral vectors, and retroviral vectors, described in more detail below.

Additional general features of adenoviruses are such that the biology of the adenovirus is characterized in detail; the adenovirus is not associated with severe human pathology; the adenovirus is extremely efficient in introducing its DNA into the host cell; the adenovirus may infect a wide variety of cells and has a broad host range; the adenovirus may be produced in large quantities with relative ease; and the adenovirus may be rendered replication defective and/or non-replicating by deletions in the early region 1 (“E1”) of the viral genome.

Adenovirus is a non-enveloped DNA virus. The genome of adenovirus is a linear double-stranded DNA molecule of approximately 36,000 base pairs (“bp”) with a 55-kDa terminal protein covalently bound to the 5′-terminus of each strand. The adenovirus DNA contains identical inverted terminal repeats (“ITRs”) of about 100 bp, with the exact length depending on the serotype. The viral origins of replication are located within the ITRs exactly at the genome ends. DNA synthesis occurs in two stages. First, replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand is single stranded and may form a “panhandle” intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may proceed from both ends of the genome simultaneously, obviating the requirement to form the panhandle structure.

During the productive infection cycle, the viral genes are expressed in two phases: the early phase, which is the period up to viral DNA replication, and the late phase, which coincides with the initiation of viral DNA replication. During the early phase, only the early gene products, encoded by regions E1, E2, E3 and E4, are expressed, which carry out a number of functions that prepare the cell for synthesis of viral structural proteins (Berk, A. J., 1986). During the late phase, the late viral gene products are expressed in addition to the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (Tooze, J., 1981).

The E1 region of adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcriptional units, the E1A and E1B genes, both of which are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the E1A gene products are to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and to transcriptionally activate the E1B gene and the other early regions (E2, E3 and E4) of the viral genome. Transfection of primary cells with the E1A gene alone may induce unlimited proliferation (immortalization), but does not result in complete transformation. However, expression of E1A, in most cases, results in induction of programmed cell death (apoptosis), and only occasionally is immortalization obtained (Jochemsen et al., 1987). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high-level expression of E1A may cause complete transformation in the absence of E1B (Roberts, B. E. et al., 1985).

The E1B encoded proteins assist E1A in redirecting the cellular functions to allow viral replication. The E1B 55 kD and E4 33 kD proteins, which form a complex that is essentially localized in the nucleus, function in inhibiting the synthesis of host proteins and in facilitating the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomitantly with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the E1B 21 kD gene product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenotype) and in an enhanced cytopathic effect (cyt-phenotype; Telling et al., 1994). The deg and cyt phenotypes are suppressed when in addition the E1A gene is mutated, indicating that these phenotypes are a function of E1A (White, E. et al., 1988). Furthermore, the E1B 21 kDa protein slows down the rate by which E1A switches on the other viral genes. It is not yet known by which mechanisms E1B 21 kD quenches these E1A dependent functions.

In contrast to, for example, retroviruses, adenoviruses do not efficiently integrate into the host cell's genome, are able to infect non-dividing cells, and are able to efficiently transfer recombinant genes in vivo (Brody et al., 1994). These features make adenoviruses attractive candidates for in vivo gene transfer of, for example, an antigen or immunogen of interest into cells, tissues or subjects in need thereof.

Adenovirus vectors containing multiple deletions are preferred to both increase the carrying capacity of the vector and reduce the likelihood of recombination to generate replication competent adenovirus (RCA). Where the adenovirus contains multiple deletions, it is not necessary that each of the deletions, if present alone, would result in a replication defective and/or non-replicating adenovirus. As long as one of the deletions renders the adenovirus replication defective or non-replicating, the additional deletions may be included for other purposes, e.g., to increase the carrying capacity of the adenovirus genome for heterologous nucleotide sequences. Preferably, more than one of the deletions prevents the expression of a functional protein and renders the adenovirus replication defective and/or non-replicating and/or attenuated. More preferably, all of the deletions are deletions that would render the adenovirus replication-defective and/or non-replicating and/or attenuated. However, the invention also encompasses adenovirus and adenovirus vectors that are replication competent and/or wild-type, i.e. comprises all of the adenoviral genes necessary for infection and replication in a subject.

Embodiments of the invention employing adenovirus recombinants may include E1-defective or deleted, or E3-defective or deleted, or E4-defective or deleted or adenovirus vectors comprising deletions of E1 and E3, or E1 and E4, or E3 and E4, or E1, E3, and E4 deleted, or the “gutless” adenovirus vector in which all viral genes are deleted. The adenovirus vectors may comprise mutations in E1, E3, or E4 genes, or deletions in these or all adenoviral genes. The E1 mutation raises the safety margin of the vector because E1-defective adenovirus mutants are said to be replication-defective and/or non-replicating in non-permissive cells, and are, at the very least, highly attenuated. The E3 mutation enhances the immunogenicity of the antigen by disrupting the mechanism whereby adenovirus down-regulates MHC class I molecules. The E4 mutation reduces the immunogenicity of the adenovirus vector by suppressing the late gene expression, thus may allow repeated re-vaccination utilizing the same vector. The present invention comprehends adenovirus vectors of any serotype or serogroup that are deleted or mutated in E1, or E3, or E4, or E1 and E3, or E1 and E4. Deletion or mutation of these adenoviral genes result in impaired or substantially complete loss of activity of these proteins.

The “gutless” adenovirus vector is another type of vector in the adenovirus vector family. Its replication requires a helper virus and a special human 293 cell line expressing both E1a and Cre, a condition that does not exist in a natural environment; the vector is deprived of all viral genes, thus the vector as a vaccine carrier is non-immunogenic and may be inoculated multiple times for re-vaccination. The “gutless” adenovirus vector also contains 36 kb space for accommodating antigen or immunogen(s) of interest, thus allowing co-delivery of a large number of antigen or immunogens into cells.

Adeno-associated virus (AAV) is a single-stranded DNA parvovirus which is endogenous to the human population. Although capable of productive infection in cells from a variety of species, AAV is a dependovirus, requiring helper functions from either adenovirus or herpes virus for its own replication. In the absence of helper functions from either of these helper viruses, AAV will infect cells, uncoat in the nucleus, and integrate its genome into the host chromosome, but will not replicate or produce new viral particles.

The genome of AAV has been cloned into bacterial plasmids and is well characterized. The viral genome consists of 4682 bases which include two terminal repeats of 145 bases each. These terminal repeats serve as origins of DNA replication for the virus. Some investigators have also proposed that they have enhancer functions. The rest of the genome is divided into two functional domains. The left portion of the genome codes for the rep functions which regulate viral DNA replication and vital gene expression. The right side of the vital genome contains the cap genes that encode the structural capsid proteins VP1, VP2 and VP3. The proteins encoded by both the rep and cap genes function in trans during productive AAV replication.

AAV is considered an ideal candidate for use as a transducing vector, and it has been used in this manner. Such AAV transducing vectors comprise sufficient cis-acting functions to replicate in the presence of adenovirus or herpes virus helper functions provided in trans. Recombinant AAV (rAAV) have been constructed in a number of laboratories and have been used to carry exogenous genes into cells of a variety of lineages. In these vectors, the AAV cap and/or rep genes are deleted from the viral genome and replaced with a DNA segment of choice. Current vectors may accommodate up to 4300 bases of inserted DNA.

To produce rAAV, plasmids containing the desired vital construct are transfected into adenovirus-infected cells. In addition, a second helper plasmid is cotransfected into these cells to provide the AAV rep and cap genes which are obligatory for replication and packaging of the recombinant viral construct. Under these conditions, the rep and cap proteins of AAV act in trans to stimulate replication and packaging of the rAAV construct. Three days after transfection, rAAV is harvested from the cells along with adenovirus. The contaminating adenovirus is then inactivated by heat treatment.

Herpes Simplex Virus 1 (HSV-1) is an enveloped, double-stranded DNA virus with a genome of 153 kb encoding more than 80 genes. Its wide host range is due to the binding of viral envelope glycoproteins to the extracellular heparin sulphate molecules found in cell membranes (WuDunn & Spear, 1989). Internalization of the virus then requires envelope glycoprotein gD and fibroblast growth factor receptor (Kaner, 1990). HSV is able to infect cells lytically or may establish latency. HSV vectors have been used to infect a wide variety of cell types (Lowenstein, 1994; Huard, 1995; Miyanohara, 1992; Liu, 1996; Goya, 1998).

There are two types of HSV vectors, called the recombinant HSV vectors and the amplicon vectors. Recombinant HSV vectors are generated by the insertion of transcription units directly into the HSV genome, through homologous recombination events. The amplicon vectors are based on plasmids bearing the transcription unit of choice, an origin of replication, and a packaging signal.

HSV vectors have the obvious advantages of a large capacity for insertion of foreign genes, the capacity to establish latency in neurons, a wide host range, and the ability to confer transgene expression to the CNS for up to 18 months (Carpenter & Stevens, 1996).

Retroviruses are enveloped single-stranded RNA viruses, which have been widely used in gene transfer protocols. Retroviruses have a diploid genome of about 7-10 kb, composed of four gene regions termed gag, pro, pol and env. These gene regions encode for structural capsid proteins, viral protease, integrase and viral reverse transcriptase, and envelope glycoproteins, respectively. The genome also has a packaging signal and cis-acting sequences, termed long-terminal repeats (LTRs), at each end, which have a role in transcriptional control and integration.

The viral vectors of the present invention are useful for the delivery of nucleic acids expressing antigens or immunogens to cells both in vitro and in vivo. In particular, the inventive vectors may be advantageously employed to deliver or transfer nucleic acids to cells, more preferably mammalian cells. Nucleic acids of interest include nucleic acids encoding peptides and proteins, preferably therapeutic (e.g., for medical or veterinary uses) or immunogenic (e.g., for vaccines) peptides or proteins.

Preferably, the codons encoding the antigen or immunogen of interest are “optimized” codons, i.e., the codons are those that appear frequently in, e.g., highly expressed genes in the subject's species, instead of those codons that are frequently used by, for example, an influenza virus. Such codon usage provides for efficient expression of the antigen or immunogen in animal cells. In other embodiments, for example, when the antigen or immunogen of interest is expressed in bacteria, yeast or another expression system, the codon usage pattern is altered to represent the codon bias for highly expressed genes in the organism in which the antigen or immunogen is being expressed. Codon usage patterns are known in the literature for highly expressed genes of many species (e.g., Nakamura et al., 1996; Wang et al., 1998; McEwan et al. 1998).

As a further alternative, the viral vectors may be used to infect a cell in culture to express a desired gene product, e.g., to produce a protein or peptide of interest. Preferably, the protein or peptide is secreted into the medium and may be purified therefrom using routine techniques known in the art. Signal peptide sequences that direct extracellular secretion of proteins are known in the art and nucleotide sequences encoding the same may be operably linked to the nucleotide sequence encoding the peptide or protein of interest by routine techniques known in the art. Alternatively, the cells may be lysed and the expressed recombinant protein may be purified from the cell lysate. Preferably, the cell is an animal cell, more preferably a mammalian cell. Also preferred are cells that are competent for transduction by particular viral vectors of interest. Such cells include PER.C6 cells, 911 cells, and HEK293 cells.

A culture medium for culturing host cells includes a medium commonly used for tissue culture, such as M199-earle base, Eagle MEM (E-MEM), Dulbecco MEM (DMEM), SC-UCM102, UP-SFM (GIBCO BRL), EX-CELL302 (Nichirei), EX-CELL293-S(Nichirei), TFBM-01 (Nichirei), ASF104, among others. Suitable culture media for specific cell types may be found at the American Type Culture Collection (ATCC) or the European Collection of Cell Cultures (ECACC). Culture media may be supplemented with amino acids such as L-glutamine, salts, anti-fungal or anti-bacterial agents such as Fungizone®, penicillin-streptomycin, animal serum, and the like. The cell culture medium may optionally be serum-free.

The present invention also relates to cell lines or transgenic animals which are capable of expressing or overexpressing LITEs or at least one agent useful in the present invention. Preferably the cell line or animal expresses or overexpresses one or more LITEs.

The transgenic animal is typically a vertebrate, more preferably a rodent, such as a rat or a mouse, but also includes other mammals such as human, goat, pig or cow etc.

Such transgenic animals are useful as animal models of disease and in screening assays for new useful compounds. By specifically expressing one or more polypeptides, as defined above, the effect of such polypeptides on the development of disease may be studied. Furthermore, therapies including gene therapy and various drugs may be tested on transgenic animals. Methods for the production of transgenic animals are known in the art. For example, there are several possible routes for the introduction of genes into embryos. These include (i) direct transfection or retroviral infection of embryonic stem cells followed by introduction of these cells into an embryo at the blastocyst stage of development; (ii) retroviral infection of early embryos; and (iii) direct microinjection of DNA into zygotes or early embryo cells. The gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art. A type of target cell for transgene introduction is the embryonic stem cell (ES). ES cells may be obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al., 1981, Nature 292:154-156; Bradley et al., 1984, Nature 309:255-258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83:9065-9069; and Robertson et al., 1986 Nature 322:445-448). Transgenes may be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction. The resultant transformed ES cells may thereafter be combined with blastocysts from a non-human animal. The introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaenisch, 1988, Science 240: 1468-1474).

LITEs may also offer valuable temporal precision in vivo. LITEs may be used to alter gene expression during a particular stage of development, for example, by repressing a particular apoptosis gene only during a particular stage of C elegans growth. LITEs may be used to time a genetic cue to a particular experimental window. For example, genes implicated in learning may be overexpressed or repressed only during the learning stimulus in a precise region of the intact rodent or primate brain. Further, LITEs may be used to induce gene expression changes only during particular stages of disease development. For example, an oncogene may be overexpressed only once a tumor reaches a particular size or metastatic stage. Conversely, proteins suspected in the development of Alzheimer's may be knocked down only at defined time points in the animal's life and within a particular brain region. Although these examples do not exhaustively list the potential applications of the LITE system, they highlight some of the areas in which LITEs may be a powerful technology.

Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.

The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.

Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co-administration or sequential administration of other agents may be desirable.

The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds identified according to this invention as the active ingredient may be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds may be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention may be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents may be administered concurrently, or they each may be administered at separately staggered times.

The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the one patient; and the particular compound thereof employed. A physician of ordinary skill may readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations may be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES Example 1

The ability to directly modulate gene expression from the endogenous mammalian genome is critical for elucidating normal gene function and disease mechanism. Advances that further refine the spatial and temporal control of gene expression within cell populations have the potential to expand the utility of gene modulation. Applicants previously developed transcription activator-like effectors (TALEs) from Xanthamonas oryze to enable the rapid design and construction of site-specific DNA binding proteins. Applicants developed a set of molecular tools for enabling light-regulated gene expression in the endogenous mammalian genome. The system consists of engineered artificial transcription factors linked to light-sensitive dimerizing protein domains from Arabidopsis thaliana. The system responds to light in the range of 450 nm-500 nm and is capable of inducing a significant increase in the expression of pluripotency factors after stimulation with light at an intensity of 6.2 mW/cm² in mammalian cells. Applicants are developing tools for the targeting of a wide range of genes. Applicants believe that a toolbox for the light-mediated control of gene expression would complement the existing optogenetic methods and may in the future help elucidate the timing-, cell type- and concentration dependent role of specific genes in the brain.

The ability to directly modulate gene expression from the endogenous mammalian genome is critical for elucidating normal gene function and disease mechanisms. Applicants present the development of a set of molecular tools for enabling light-regulated gene expression in the endogenous mammalian genome. This system consists of a transcription activator like effector (TALE) and the activation domain VP64 linked to the light-sensitive dimerizing protein domains cryptochrome 2 (CRY2) and CIB1 from Arabidopsis thaliana. Applicants show that blue-light stimulation of HEK293FT and Neuro-2a cells transfected with these LITE constructs designed to target the promoter region of KLF4 and Neurog2 results in a significant increase in target expression, demonstrating the functionality of TALE-based optical gene expression modulation technology.

FIG. 2 shows transcription activator like effectors (TALEs). TALEs consist of 34 aa repeats (SEQ ID NO:1) at the core of their sequence. Each repeat corresponds to a base in the target DNA that is bound by the TALE. Repeats differ only by 2 variable amino acids at positions 12 and 13. The code of this correspondence has been elucidated (Boch, J et al., Science, 2009 and Moscou, M et al., Science, 2009) and is shown in this figure. One example of a binding site is shown as SEQ ID NO: 2. Applicants developed a method for the synthesis of designer TALEs incorporating this code and capable of binding a sequence of choice within the genome (Zhang, F et al., Nature Biotechnology, 2011).

FIG. 3 depicts a design of a LITE: TALE/Cryptochrome transcriptional activation. Each LITE is a two-component system which may comprise a TALE fused to CRY2 and the cryptochrome binding partner CIB1 fused to VP64, a transcription activator. In the inactive state, the TALE localizes its fused CRY2 domain to the promoter region of the gene of interest. At this point, CIB1 is unable to bind CRY2, leaving the CIB1-VP64 unbound in the nuclear space. Upon stimulation with 488 nm (blue) light, CRY2 undergoes a conformational change, revealing its CIB1 binding site (Liu, H et al., Science, 2008). Rapid binding of CIB1 results in recruitment of the fused VP64 domain, which induces transcription of the target gene.

Example 2

Normal gene expression is a dynamic process with carefully orchestrated temporal and spatial components, the precision of which are necessary for normal development, homeostasis, and advancement of the organism. In turn, the dysregulation of required gene expression patterns, either by increased, decreased, or altered function of a gene or set of genes, has been linked to a wide array of pathologies. Technologies capable of modulating gene expression in a spatiotemporally precise fashion will enable the elucidation of the genetic cues responsible for normal biological processes and disease mechanisms. To address this technological need, Applicants developed light-inducible transcriptional effectors (LITEs), which provide light-mediated control of endogenous gene expression.

Inducible gene expression systems have typically been designed to allow for chemically inducible activation of an inserted open reading frame or shRNA sequence, resulting in gene overexpression or repression, respectively. Disadvantages of using open reading frames for overexpression include loss of splice variation and limitation of gene size. Gene repression via RNA interference, despite its transformative power in human biology, may be hindered by complicated off-target effects. Certain inducible systems including estrogen, ecdysone, and FKBP12/FRAP based systems are known to activate off-target endogenous genes. The potentially deleterious effects of long-term antibiotic treatment may complicate the use of tetracycline transactivator (TET) based systems. In vivo, the temporal precision of these chemically inducible systems is dependent upon the kinetics of inducing agent uptake and elimination. Further, because inducing agents are generally delivered systemically, the spatial precision of such systems is bounded by the precision of exogenous vector delivery.

In response to these limitations, LITEs are designed to modulate expression of individual endogenous genes in a temporally and spatially precise manner. Each LITE is a two component system consisting of a customized DNA-binding transcription activator like effector (TALE) protein, a light-responsive cryptochrome heterodimer from Arabadopsis thaliana, and a transcriptional activation/repression domain. The TALE is designed to bind to the promoter sequence of the gene of interest. The TALE protein is fused to one half of the cryptochrome heterodimer (cryptochrome-2 or CIB1), while the remaining cryptochrome partner is fused to a transcriptional effector domain. Effector domains may be either activators, such as VP16, VP64, or p65, or repressors, such as KRAB, EnR, or SID. In a LITE's unstimulated state, the TALE-cryptochrome2 protein localizes to the promoter of the gene of interest, but is not bound to the CIB1-effector protein. Upon stimulation of a LITE with blue spectrum light, cryptochrome-2 becomes activated, undergoes a conformational change, and reveals its binding domain. CIB1, in turn, binds to cryptochrome-2 resulting in localization of the effector domain to the promoter region of the gene of interest and initiating gene overexpression or silencing.

Gene targeting in a LITE is achieved via the specificity of customized TALE DNA binding proteins. A target sequence in the promoter region of the gene of interest is selected and a TALE customized to this sequence is designed. The central portion of the TALE consists of tandem repeats 34 amino acids in length. Although the sequences of these repeats are nearly identical, the 12th and 13th amino acids (termed repeat variable diresidues) of each repeat vary, determining the nucleotide-binding specificity of each repeat. Thus, by synthesizing a construct with the appropriate ordering of TALE monomer repeats, a DNA binding protein specific to the target promoter sequence is created.

Light responsiveness of a LITE is achieved via the activation and binding of cryptochrome-2 and CIB1. As mentioned above, blue light stimulation induces an activating conformational change in cryptochrome-2, resulting in recruitment of its binding partner CIB1. This binding is fast and reversible, achieving saturation in <15 sec following pulsed stimulation and returning to baseline <15 min after the end of stimulation. These rapid binding kinetics result in a LITE system temporally bound only by the speed of transcription/translation and transcript/protein degradation, rather than uptake and clearance of inducing agents. Cryptochrome-2 activation is also highly sensitive, allowing for the use of low light intensity stimulation and mitigating the risks of phototoxicity. Further, in a context such as the intact mammalian brain, variable light intensity may be used to control the size of a LITE stimulated region, allowing for greater precision than vector delivery alone may offer.

The modularity of the LITE system allows for any number of effector domains to be employed for transcriptional modulation. Thus, activator and repressor domains may be selected on the basis of species, strength, mechanism, duration, size, or any number of other parameters.

Applicants next present two prototypical manifestations of the LITE system. The first example is a LITE designed to activate transcription of the mouse gene NEUROG2. The sequence TGAATGATGATAATACGA (SEQ ID NO:149), located in the upstream promoter region of mouse NEUROG2, was selected as the target and a TALE was designed and synthesized to match this sequence. The TALE sequence was linked to the sequence for cryptochrome-2 via a nuclear localization signal (amino acids: SPKKKRKVEAS; SEQ ID NO: 150) to facilitate transport of the protein from the cytosol to the nuclear space. A second vector was synthesized comprising the CIB1 domain linked to the transcriptional activator domain VP64 using the same nuclear localization signal. This second vector, also a GFP sequence, is separated from the CIB1-VP64 fusion sequence by a 2A translational skip signal. Expression of each construct was driven by a ubiquitous, constitutive promoter (CMV or EF1-α). Mouse neuroblastoma cells from the Neuro 2A cell line were co-transfected with the two vectors. After incubation to allow for vector expression, samples were stimulated by periodic pulsed blue light from an array of 488 nm LEDs. Unstimulated co-transfected samples and samples transfected only with the fluorescent reporter YFP were used as controls. At the end of each experiment, mRNA was purified from the samples analyzed via qPCR.

Truncated versions of cryptochrome-2 and CIB1 were cloned and tested in combination with the full-length versions of cryptochrome-2 and CIB1 in order to determine the effectiveness of each heterodimer pair. The combination of the CRY2PHR domain, consisting of the conserved photoresponsive region of the cryptochrome-2 protein, and the full-length version of CIB1 resulted in the highest upregulation of Neurog2 mRNA levels (˜22 fold over YFP samples and ˜7 fold over unstimulated co-transfected samples). The combination of full-length cryptochrome-2 (CRY2) with full-length CIB1 resulted in a lower absolute activation level (˜4.6 fold over YFP), but also a lower baseline activation (˜1.6 fold over YFP for unstimulated co-transfected samples). These cryptochrome protein pairings may be selected for particular uses depending on absolute level of induction required and the necessity to minimize baseline “leakiness” of the LITE system.

Speed of activation and reversibility are critical design parameters for the LITE system. To characterize the kinetics of the LITE system, constructs consisting of the Neurog2 TALE-CRY2PHR and CIB1-VP64 version of the system were tested to determine its activation and inactivation speed. Samples were stimulated for as little as 0.5 h to as long as 24 h before extraction. Upregulation of Neurog2 expression was observed at the shortest, 0.5 h, time point (˜5 fold vs YFP samples). Neurog2 expression peaked at 12 h of stimulation (˜19 fold vs YFP samples). Inactivation kinetics were analyzed by stimulating co-transfected samples for 6 h, at which time stimulation was stopped, and samples were kept in culture for 0 to 12 h to allow for mRNA degradation. Neurog2 mRNA levels peaked at 0.5 h after the end of stimulation (˜16 fold vs. YFP samples), after which the levels degraded with an ˜3 h half-life before returning to near baseline levels by 12 h.

The second prototypical example is a LITE designed to activate transcription of the human gene KLF4. The sequence TTCTTACTTATAAC (SEQ ID NO: 167), located in the upstream promoter region of human KLF4, was selected as the target and a TALE was designed and synthesized to match this sequence. The TALE sequence was linked to the sequence for CRY2PHR via a nuclear localization signal (amino acids: SPKKKRKVEAS; SEQ ID NO: 150). The identical CIB1-VP64 activator protein described above was also used in this manifestation of the LITE system. Human embryonal kidney cells from the HEK293FT cell line were co-transfected with the two vectors. After incubation to allow for vector expression, samples were stimulated by periodic pulsed blue light from an array of 488 nm LEDs. Unstimulated co-transfected samples and samples transfected only with the fluorescent reporter YFP were used as controls. At the end of each experiment, mRNA was purified from the samples analyzed via qPCR.

The light-intensity response of the LITE system was tested by stimulating samples with increased light power (0-9 mW/cm2). Upregulation of KLF4 mRNA levels was observed for stimulation as low as 0.2 mW/cm2. KLF4 upregulation became saturated at 5 mW/cm2 (2.3 fold vs. YFP samples). Cell viability tests were also performed for powers up to 9 mW/cm2 and showed >98% cell viability. Similarly, the KLF4 LITE response to varying duty cycles of stimulation was tested (1.6-100%). No difference in KLF4 activation was observed between different duty cycles indicating that a stimulation paradigm of as low as 0.25 sec every 15 sec should result in maximal activation.

There are potential applications for which LITEs represent an advantageous choice for gene expression control. There exist a number of in vitro applications for which LITEs are particularly attractive. In all these cases, LITEs have the advantage of inducing endogenous gene expression with the potential for correct splice variant expression.

Because LITE activation is photoinducible, spatially defined light patterns, created via masking or rasterized laser scanning, may be used to alter expression levels in a confined subset of cells. For example, by overexpressing or silencing an intercellular signaling molecule only in a spatially constrained set of cells, the response of nearby cells relative to their distance from the stimulation site may help elucidate the spatial characteristics of cell non-autonomous processes. Additionally, recent advances in cell reprogramming biology have shown that overexpression of sets of transcription factors may be utilized to transform one cell type, such as fibroblasts, into another cell type, such as neurons or cardiomyocytes. Further, the correct spatial distribution of cell types within tissues is critical for proper organotypic function. Overexpression of reprogramming factors using LITEs may be employed to reprogram multiple cell lineages in a spatially precise manner for tissue engineering applications.

The rapid transcriptional response and endogenous targeting of LITEs make for an ideal system for the study of transcriptional dynamics. For example, LITEs may be used to study the dynamics of mRNA splice variant production upon induced expression of a target gene. On the other end of the transcription cycle, mRNA degradation studies are often performed in response to a strong extracellular stimulus, causing expression level changes in a plethora of genes. LITEs may be utilized to reversibly induce transcription of an endogenous target, after which point stimulation may be stopped and the degradation kinetics of the unique target may be tracked.

The temporal precision of LITEs may provide the power to time genetic regulation in concert with experimental interventions. For example, targets with suspected involvement in long-term potentiation (LTP) may be modulated in organotypic or dissociated neuronal cultures, but only during stimulus to induce LTP, so as to avoid interfering with the normal development of the cells. Similarly, in cellular models exhibiting disease phenotypes, targets suspected to be involved in the effectiveness of a particular therapy may be modulated only during treatment. Conversely, genetic targets may be modulated only during a pathological stimulus. Any number of experiments in which timing of genetic cues to external experimental stimuli is of relevance may potentially benefit from the utility of LITE modulation.

The in vivo context offers equally rich opportunities for the use of LITEs to control gene expression. As mentioned above, photoinducibility provides the potential for previously unachievable spatial precision. Taking advantage of the development of optrode technology, a stimulating fiber optic lead may be placed in a precise brain region. Stimulation region size may then be tuned by light intensity. This may be done in conjunction with the delivery of LITEs via viral vectors, or, if transgenic LITE animals were to be made available, may eliminate the use of viruses while still allowing for the modulation of gene expression in precise brain regions. LITEs may be used in a transparent organism, such as an immobilized zebrafish, to allow for extremely precise laser induced local gene expression changes.

LITEs may also offer valuable temporal precision in vivo. LITEs may be used to alter gene expression during a particular stage of development, for example, by repressing a particular apoptosis gene only during a particular stage of C elegans growth. LITEs may be used to time a genetic cue to a particular experimental window. For example, genes implicated in learning may be overexpressed or repressed only during the learning stimulus in a precise region of the intact rodent or primate brain. Further, LITEs may be used to induce gene expression changes only during particular stages of disease development. For example, an oncogene may be overexpressed only once a tumor reaches a particular size or metastatic stage. Conversely, proteins suspected in the development of Alzheimer's may be knocked down only at defined time points in the animal's life and within a particular brain region. Although these examples do not exhaustively list the potential applications of the LITE system, they highlight some of the areas in which LITEs may be a powerful technology.

Example 3 Development of Mammalian TALE ToolBox

Customized TALEs may be used for a wide variety of genome engineering applications, including transcriptional modulation and genome editing. Here, Applicants describe a toolbox for rapid construction of custom TALE transcription factors (TALE-TFs) and nucleases (TALENs) using a hierarchical ligation procedure. This toolbox facilitates affordable and rapid construction of custom TALE-TFs and TALENs within 1 week and may be easily scaled up to construct TALEs for multiple targets in parallel. Applicants also provide details for testing the activity in mammalian cells of custom TALE-TFs and TALENs using quantitative reverse-transcription PCR and Surveyor nuclease, respectively. The TALE toolbox will enable a broad range of biological applications.

TALEs are natural bacterial effector proteins used by Xanthomonas sp. to modulate gene transcription in host plants to facilitate bacterial colonization (7, 8). The central region of the protein contains tandem repeats of 34-aa sequences (termed monomers; e.g., SEQ ID NO: 1) that are required for DNA recognition and binding (9, 10, 11, 12) (FIG. 8). Naturally occurring TALEs have been found to have a variable number of monomers, ranging from 1.5 to 33.5 (7). Although the sequence of each monomer is highly conserved, they differ primarily in two positions termed the repeat variable diresidues (RVDs, 12th and 13th positions). Recent reports have found that the identity of these two residues determines the nucleotide-binding specificity of each TALE repeat and that a simple cipher specifies the target base of each RVD (NI=A, HD=C, NG=T, NN=G or A) (1, 2). Thus, each monomer targets one nucleotide and the linear sequence of monomers in a TALE specifies the target DNA sequence in the 5′ to 3′ orientation. The natural TALE-binding sites within plant genomes always begin with a thymine (1, 2), which is presumably specified by a cryptic signal within the nonrepetitive N terminus of TALEs. The tandem repeat DNA-binding domain always ends with a half-length repeat (0.5 repeat, FIG. 8). Therefore, the length of the DNA sequence being targeted is equal to the number of full repeat monomers plus two.

Applicants have further improved the TALE assembly system with a few optimizations, including maximizing the dissimilarity of ligation adaptors to minimize misligations and combining separate digest and ligation steps into single Golden Gate (13, 14, 15) reactions. Briefly, each nucleotide-specific monomer sequence is amplified with ligation adaptors that uniquely specify the monomer position within the TALE tandem repeats. Once this monomer library is produced, it may conveniently be reused for the assembly of many TALEs. For each TALE desired, the appropriate monomers are first ligated into hexamers, which are then amplified via PCR. Then, a second Golden Gate digestion-ligation with the appropriate TALE cloning backbone (FIG. 8) yields a fully assembled, sequence-specific TALE. The backbone contains a ccdB negative selection cassette flanked by the TALE N and C termini, which is replaced by the tandem repeat DNA-binding domain when the TALE has been successfully constructed. ccdB selects against cells transformed with an empty backbone, thereby yielding clones with tandem repeats inserted (5).

Assemblies of monomeric DNA-binding domains may be inserted into the appropriate TALE-TF or TALEN cloning backbones to construct customized TALE-TFs and TALENs. TALE-TFs are constructed by replacing the natural activation domain within the TALE C terminus with the synthetic transcription activation domain VP64 (3; FIG. 8).

REFERENCES

-   1. Boch, J. et al. Breaking the code of DNA binding specificity of     TAL-type III effectors. Science 326, 1509-1512 (2009). -   2. Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA     recognition by TAL effectors. Science 326, 1501 (2009). -   3. Zhang, F. et al. Efficient construction of sequence-specific TAL     effectors for modulating mammalian transcription. Nat. Biotechnol.     29, 149-153 (2011). -   4. Miller, J. C. et al. A TALE nuclease architecture for efficient     genome editing. Nat. Biotechnol. 29, 143-148 (2011). -   5. Cermak, T. et al. Efficient design and assembly of custom TALEN     and other TAL effector-based constructs for DNA targeting. Nucleic     Acids Res. 39, e82 (2011). -   6. Hockemeyer, D. et al. Genetic engineering of human pluripotent     cells using TALE nucleases. Nat. Biotechnol. 29, 731-734 (2011). -   7. Boch, J. & Bonas, U. Xanthomonas AvrBs3 family-type III     effectors: discovery and function. Annu. Rev. Phytopathol. 48,     419-436 (2010). -   8. Bogdanove, A. J., Schornack, S. & Lahaye, T. TAL effectors:     finding plant genes for disease and defense. Curr. Opin. Plant Biol.     13, 394-401 (2010). -   9. Romer, P. et al. Plant pathogen recognition mediated by promoter     activation of the pepper Bs3 resistance gene. Science 318, 645-648     (2007). -   10. Kay, S., Hahn, S., Marois, E., Hause, G. & Bonas, U. A bacterial     effector acts as a plant transcription factor and induces a cell     size regulator. Science 318, 648-651 (2007). -   11. Kay, S., Hahn, S., Marois, E., Wieduwild, R. & Bonas, U.     Detailed analysis of the DNA recognition motifs of the Xanthomonas     type III effectors AvrBs3 and AvrBs3Deltarep16. Plant J. 59, 859-871     (2009). -   12. Romer, P. et al. Recognition of AvrBs3-like proteins is mediated     by specific binding to promoters of matching pepper Bs3 alleles.     Plant Physiol. 150, 1697-1712 (2009). -   13. Engler, C., Kandzia, R. & Marillonnet, S. A one pot, one step,     precision cloning method with high throughput capability. PLoS ONE     3, e3647 (2008). -   14. Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden     gate shuffling: a one-pot DNA shuffling method based on type IIs     restriction enzymes. PLoS ONE 4, e5553 (2009). -   15. Weber, E., Engler, C., Gruetzner, R., Werner, S. &     Marillonnet, S. A modular cloning system for standardized assembly     of multigene constructs. PLoS ONE 6, e16765 (2011). -   16. Huertas, P. DNA resection in eukaryotes: deciding how to fix the     break. Nat. Struct. Mol. Biol. 17, 11-16 (2010).

Example 4

FIG. 17 depicts an effect of cryptochrome2 heterodimer orientation on LITE functionality. Two versions of the Neurogenin 2 (Neurog2) LITE were synthesized to investigate the effects of cryptochrome 2 photolyase homology region (CRY2PHR)/calcium and integrin-binding protein 1 (CIB1) dimer orientation. In one version, the CIB1 domain was fused to the C-terminus of the TALE (Neurog2) domain, while the CRY2PHR domain was fused to the N-terminus of the VP64 domain. In the converse version, the CRY2PHR domain was fused to the C-terminus of the TALE (Neurog2) domain, while the CIB1 domain was fused to the N-terminus of the VP64 domain. Each set of plasmids were transfected in Neuro2a cells and stimulated (466 nm, 5 mW/cm², 1 sec pulse per 15 sec, 12 h) before harvesting for qPCR analysis. Stimulated LITE and unstimulated LITE Neurog2 expression levels were normalized to Neurog2 levels from stimulated GFP control samples. The TALE-CRY2PHR/CIB1-VP64 LITE exhibited elevated basal activity and higher light induced Neurog2 expression, and suggested its suitability for situations in which higher absolute activation is required. Although the relative light inducible activity of the TALE-CIB1/CRY2PHR-VP64 LITE was lower that its counterpart, the lower basal activity suggested its utility in applications requiring minimal baseline activation. Further, the TALE-CIB1 construct was smaller in size, compared to the TALE-CRY2PHR construct, a potential advantage for applications such as viral packaging.

FIG. 18 depicts metabotropic glutamate receptor 2 (mGlur2) LITE activity in mouse cortical neuron culture. A mGluR2 targeting LITE was constructed via the plasmids pAAV-human Synapsin I promoter (hSyn)-HA-TALE(mGluR2)-CIB1 and pAAV-hSyn-CRY2PHR-VP64-2A-GFP. These fusion constructs were then packaged into adeno associated viral vectors (AAV). Additionally, AAV carrying hSyn-TALE-VP64-2A-GFP and GFP only were produced. Embryonic mouse (E16) cortical cultures were plated on Poly-L-lysine coated 24 well plates. After 5 days in vitro neural cultures were co-transduced with a mixture of TALE(mGluR2)-CIB1 and CRY2PHR-VP64 AAV stocks. Control samples were transduced with either TALE(mGluR2)-VP64 AAV or GFP AAV. 6 days after AAV transduction, experimental samples were stimulated using either of two light pulsing paradigms: 0.5 s per min and 0.25 sec per 30 sec. Neurons were stimulated for 24 h and harvested for qPCR analysis. All mGluR2 expression levels were normalized to the respective stimulated GFP control. The data suggested that the LITE system could be used to induce the light-dependent activation of a target gene in primary neuron cultures in vitro.

FIG. 19 depicts transduction of primary mouse neurons with LITE AAV vectors. Primary mouse cortical neuron cultures were co-transduced at 5 days in vitro with AAV vectors encoding hSyn-CRY2PHR-VP64-2A-GFP and hSyn-HA-TALE-CIB1, the two components of the LITE system. Left panel: at 6 days after transduction, neural cultures exhibited high expression of GFP from the hSyn-CRY2PHR-VP64-2A-GFP vector. Right panel: Co-transduced neuron cultures were fixed and stained with an antibody specific to the HA epitope on the N-terminus of the TALE domain in hSyn-HA-TALE-CIB1. Red signal indicated HA expression, with particularly strong nuclear signal (DNA stained by DAPI in blue channel). Together these images suggested that the expression of each LITE component could be achieved in primary mouse neuron cultures. (scale bars=50 um).

FIG. 20 depicts expression of a LITE component in vivo. An AAV vector of serotype 1/2 carrying hSyn-CRY2PHR-VP64 was produced via transfection of HEK293FT cells and purified via heparin column binding. The vector was concentrated for injection into the intact mouse brain. 1 uL of purified AAV stock was injected into the hippocampus and infralimbic cortex of an 8 week old male C57BL/6 mouse by steroeotaxic surgery and injection. 7 days after in vivo transduction, the mouse was euthanized and the brain tissue was fixed by paraformaldehyde perfusion. Slices of the brain were prepared on a vibratome and mounted for imaging. Strong and widespread GFP signals in the hippocampus and infralimbic cortex suggested efficient transduction and high expression of the LITE component CRY2PHR-VP64.

Example 5 Multiplex Genome Engineering Using CRISPR/Cas Systems

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. Applicants engineered two different type II CRISPR systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Finally, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the CRISPR technology.

Prokaryotic CRISPR adaptive immune systems can be reconstituted and engineered to mediate multiplex genome editing in eukaryote cells, advantageously mammalian cells.

Precise and efficient genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements. Although genome-editing technologies such as designer zinc fingers (ZFs) (1-4), transcription activator-like effectors (TALEs) (4-10), and homing meganucleases (11) have begun to enable targeted genome modifications, there remains a need for new technologies that are scalable, affordable, and easy to engineer. Here, Applicants report the development of a new class of precision genome engineering tools based on the RNA-guided Cas9 nuclease (12-14) from the type II prokaryotic CRISPR adaptive immune system (15-18).

The Streptococcus pyogenes SF370 type II CRISPR locus consists of four genes, including the Cas9 nuclease, as well as two non-coding RNAs: tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs) (FIG. 27) (19). Applicants sought to harness this prokaryotic RNA-programmable nuclease system to introduce targeted double stranded breaks (DSBs) in mammalian chromosomes through heterologous expression of the key components. It has been previously shown that expression of tracrRNA, pre-crRNA, host factor RNase III, and Cas9 nuclease are necessary and sufficient for cleavage of DNA in vitro (12, 13) and in prokaryotic cells (20, 21). Applicants codon optimized the S. pyogenes Cas9 (SpCas9) and RNase III (SpRNase III) and attached nuclear localization signals (NLS) to ensure nuclear compartmentalization in mammalian cells. Expression of these constructs in human 293FT cells revealed that two NLSs are required for targeting SpCas9 to the nucleus (FIG. 23A). To reconstitute the non-coding RNA components of CRISPR, Applicants expressed an 89-nucleotide (nt) tracrRNA (FIG. 28) under the RNA polymerase III U6 promoter (FIG. 23B). Similarly, Applicants used the U6 promoter to drive the expression of a pre-crRNA array comprising a single guide spacer flanked by DRs (FIG. 23B). Applicants designed an initial spacer to target a 30-basepair (bp) site (protospacer) in the human EMX1 locus that precedes an NGG, the requisite protospacer adjacent motif (PAM) (FIG. 23C and FIG. 27) (22, 23).

To test whether heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA, and pre-crRNA) can achieve targeted cleavage of mammalian chromosomes, Applicants transfected 293FT cells with different combinations of CRISPR components. Since DSBs in mammalian DNA are partially repaired by the indel-forming non-homologous end joining (NHEJ) pathway, Applicants used the SURVEYOR assay to detect endogenous target cleavage (FIG. 23D). Co-transfection of all four required CRISPR components resulted in efficient cleavage of the protospacer (FIG. 23D), which is subsequently verified by Sanger sequencing (FIG. 23E). Removing any of the remaining RNA or Cas9 components abolished the genome cleavage activity of the CRISPR system (FIG. 23D). These results define a minimal three-component system for efficient CRISPR-mediated genome modification in mammalian cells.

Example 6 Optical Control of Endogenous Mammalian Transcription

The ability to directly modulate transcription of the endogenous mammalian genome is critical for elucidating normal gene function and disease mechanisms. Here, Applicants describe the development of Light-Inducible Transcriptional Effectors (LITEs), a two-component system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs can be engineered and delivered to mediate positive and negative regulation of endogenous mammalian gene expression in a reversible manner, and changes in mRNA levels occur within minutes after optical illumination. Applicants have applied this system in cell lines, primary mouse neurons, as well as in the brain of awake, behaving mice in vivo.

An ideal optogenetic approach for controlling endogenous gene transcription would be readily generalizable to target any gene locus, would not require manipulation of the endogenous genomic sequence, would not depend on the addition of exogenous chemical co-factors, and would exhibit fast and reversible kinetics. The DNA-binding domain of transcription activator-like effectors (TALEs) (13, 14) from Xanthomonas sp. can be easily customized to bind specific DNA sequences in mammalian cells (15-17). TALE DNA-binding domains are modular and can be fused with a variety of effector domains, including nucleases, transcriptional activators, and transcriptional repressors to edit or modulate endogenous mammalian genomic loci (15-18). Applicants sought to combine TALEs with light-sensitive proteins to create a suite of tools for enabling spatiotemporally precise control of endogenous gene transcription.

Here, Applicants report the development of Light-Inducible Transcriptional Effectors (LITEs), a two-component system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana (8, 19). LITEs can be engineered to mediate positive and negative regulation of endogenous mammalian gene expression in a reversible manner, and changes in transcript levels occur within minutes after stimulation. Like other optogenetic tools, LITEs can be packaged into viral vectors and genetically targeted to probe gene function within specific cell populations. Applicants demonstrate the application of this system in primary neurons as well as in the mouse brain in vivo.

In the design of the LITE system, Applicants sought to use light-inducible heterodimeric proteins to mediate the recruitment of transcriptional effector domains to a TALE targeted to an endogenous genomic locus. While several plant-based light-sensitive proteins have been developed for mammalian applications, some suffer from slow or irreversible kinetics while others depend on the supplementation of exogenous co-factors that are not present in mammalian cells (5, 6, 9). The Arabidopsis thaliana cryptochrome 2 (CRY2) was previously shown to employ flavin adenine dinucleotide—an abundant biomolecule in mammalian cells—as its light-sensing chromophore¹⁹. The flavin chromophore is reduced upon photoexcitation with blue light (peak ˜450 nm), triggering a conformational change in CRY2 that allows dimerization with its interacting protein partner CIB1¹⁹. The dimerization between CRY2 and CIB1 occurs within seconds and is reversible within a few minutes following withdrawal of light illumination⁸. Based on these properties, Applicants selected CRY2 and CIB1 as light-sensing components for constructing LITEs.

Manipulating endogenous gene expression presents various challenges, as the rate of expression depends on many factors, including regulatory elements, mRNA processing, and transcript stability (22, 23). Applicants sought to investigate the feasibility of using the system to modulate endogenous gene expression in primary neurons and the intact brain. To this end, Applicants pursued viral transduction as an effective method for TALE and LITE gene delivery into neurons. However, lentiviral delivery can compromise TALE integrity due to recombination of the tandem repeat DNA-binding domains during reverse transcription (26). To overcome this challenge, Applicants developed an adeno-associated virus (AAV)-based vector for the delivery of TALE genes and efficient process for AAV production (FIGS. 37A-B, FIG. 42, and Example 7). AAV has an ssDNA-based genome and is therefore less susceptible to recombination (27-29).

AAV1/2 (serotype AAV1/2, i.e., hybrid or mosaic AAV1/AAV2 capsid AAV) heparin purified concentrated virus protocol

Media: D10+HEPES

500 ml bottle DMEM high glucose+Glutamax (GIBCO) 50 ml Hyclone FBS (heat-inactivated) (Thermo Fischer) 5.5 ml HEPES solution (1M, GIBCO) Cells: low passage HEK293FT (passage <10 at time of virus production, thaw new cells of passage 2-4 for virus production, grow up for 3-5 passages)

Transfection Reagent: Polyethylenimine (PEI) “Max”

Dissolve 50 mg PEI “Max” in 50 ml sterile Ultrapure H₂O

Adjust pH to 7.1

Filter with 0.22 um fliptop filter Seal tube and wrap with parafilm Freeze aliquots at −20° C. (for storage, can also be used immediately)

Cell Culture

Culture low passage HEK293FT in D10+HEPES Passage everyday between 1:2 and 1:2.5 Advantageously do not allow cells to reach more than 85% confluency

For T75

Warm 10 ml HBSS (—Mg2+, —Ca2+, GIBCO)+1 ml TrypLE Express (GIBCO) per flask to 37° C. (Waterbath)

Aspirate media fully

Add 10 ml warm HBSS gently (to wash out media completely)

Add 1 ml TrypLE per Flask

Place flask in incubator (37° C.) for 1 min

Rock flask to detach cells

Add 9 ml D10+HEPES media (37° C.)

Pipette up and down 5 times to generate single cell suspension

Split at 1:2-1:2.5 (12 ml media for T75) ratio (if cells are growing more slowly, discard and thaw a new batch, they are not in optimal growth)

transfer to T225 as soon as enough cells are present (for ease of handling large amounts of cells)

AAV Production (5*15 cm Dish Scale Per Construct):

Plate 10 million cells in 21.5 ml media into a 15 cm dish Incubate for 18-22 hours at 37° C. Transfection is ideal at 80% confluence

Per Plate

Prewarm 22 ml media (D10+HEPES) Prepare Tube with DNA Mixture (Use Endofree Maxiprep DNA): 5.2 ug vector of interest plasmid 4.35 ug AAV 1 serotype plasmid 4.35 ug AAV 2 serotype plasmid 10.4 ug pDF6 plasmid (adenovirus helper genes)

→Vortex to mix

Add 434 uL DMEM (no serum!) Add 130 ul PEI solution Vortex 5-10 seconds Add DNA/DMEM/PEI mixture to prewarmed media →Vortex briefly to mix Replace media in 15 cm dish with DNA/DMEM/PEI mixture →Return to 37° C. incubator →Incubate 48 h before harvesting (make sure medium isn't turning too acidic)

Virus Harvest:

1. aspirate media carefully from 15 cm dish dishes (advantageously do not dislodge cells) 2. Add 25 ml RT DPBS (Invitrogen) to each plate and gently remove cells with a cell scraper. Collect suspension in 50 ml tubes. 3. Pellet cells at 800×g for 10 minutes. 4. Discard supernatant →pause point: freeze cell pellet at −80 C if desired 5. resuspend pellet in 150 mM NaCl, 20 mM Tris pH 8.0, use 10 ml per tissue culture plate. 6. Prepare a fresh solution of 10% sodium deoxycholate in dH2O. Add 1.25 ml of this per tissue culture plate for a final concentration of 0.5%. Add benzonase nuclease to a final concentration of 50 units per ml. Mix tube thoroughly. 7. Incubate at 37° C. for 1 hour (Waterbath). 8. Remove cellular debris by centrifuging at 3000×g for 15 mins. Transfer to fresh 50 ml tube and ensure all cell debris has been removed to prevent blocking of heparin columns.

Heparin Column Purification of AAV1/2:

1. Set up HiTrap heparin columns using a peristaltic pump so that solutions flow through the column at 1 ml per minute. It is important to ensure no air bubbles are introduced into the heparin column. 2. Equilibrate the column with 10 ml 150 mM NaCl, 20 mM Tris, pH 8.0 using the peristaltic pump. 3. Binding of virus: Apply 50 ml virus solution to column and allow to flow through. 4. Wash step 1: column with 20 ml 100 mM NaCl, 20 mM Tris, pH 8.0. (using the peristaltic pump) 5. Wash step 2: Using a 3 ml or 5 ml syringe continue to wash the column with 1 ml 200 mM NaCl, 20 mM Tris, pH 8.0, followed by 1 ml 300 mM NaCl, 20 mM Tris, pH 8.0. →Discard the flow-through. (prepare the syringes with different buffers during the 50 min flow through of virus solution above) 6. Elution Using 5 ml syringes and gentle pressure (flow rate of <1 ml/min) elute the virus from the column by applying:

1.5 ml 400 mM NaCl, 20 mM Tris, pH 8.0 3.0 ml 450 mM NaCl, 20 mM Tris, pH 8.0 1.5 ml 500 mM NaCl, 20 mM Tris, pH 8.0

Collect these in a 15 ml centrifuge tube.

Concentration of AAV1/2:

1. Concentration step 1: Concentrate the eluted virus using Amicon ultra 15 ml centrifugal filter units with a 100,000 molecular weight cutoff. Load column eluate into the concentrator and centrifuge at 2000×g for 2 minutes (at room temperature. Check concentrated volume—it should be approximately 500 μl. If necessary, centrifuge in 1 min intervals until correct volume is reached. 2. buffer exchange: Add 1 ml sterile DPBS to filter unit, centrifuge in 1 min intervals until correct volume (500 ul) is reached. 3. Concentration step 2: Add 500 ul concentrate to an Amicon Ultra 0.5 ml 100K filter unit. Centrifuge at 6000 g for 2 min. Check concentrated volume—it should be approximately 100 μl. If necessary, centrifuge in 1 min intervals until correct volume is reached. 4. Recovery: Invert filter insert and insert into fresh collection tube. Centrifuge at 1000 g for 2 min. →Aliquot and freeze at −80° C. →1 ul is typically required per injection site, small aliquots (e.g. 5 ul) are therefore recommended (avoid freeze-thaw of virus). →determine DNaseI-resistant GC particle titer using qPCR (see separate protocol)

Materials

Amicon Ultra, 0.5 ml, 100K; MILLIPORE; UFC510024 Amicon Ultra, 15 ml, 100K; MILLIPORE; UFC910024

Benzonase nuclease; Sigma-Aldrich, E1014 HiTrap Heparin cartridge; Sigma-Aldrich; 54836 Sodium deoxycholate; Sigma-Aldrich; D5670

AAV1 Supernatant Production Protocol

Media: D10+HEPES

500 ml bottle DMEM high glucose+Glutamax (Invitrogen) 50 ml Hyclone FBS (heat-inactivated) (Thermo Fischer) 5.5 ml HEPES solution (1M, GIBCO)

Cells: low passage HEK293FT (passage <10 at time of virus production)

Thaw new cells of passage 2-4 for virus production, grow up for 2-5 passages Transfection reagent: Polyethylenimine (PEI) “Max” Dissolve 50 mg PEI “Max” in 50 ml sterile Ultrapure H₂O

Adjust pH to 7.1

Filter with 0.22 um fliptop filter Seal tube and wrap with parafilm Freeze aliquots at −20° C. (for storage, can also be used immediately)

Cell Culture

Culture low passage HEK293FT in D10+HEPES Passage everyday between 1:2 and 1:2.5 Advantageously do let cells reach more than 85% confluency

For T75

Warm 10 ml HBSS (—Mg2+, —Ca2+, GIBCO)+1 ml TrypLE Express (GIBCO) per flask to 37° C. (Waterbath)

Aspirate media fully

Add 10 ml warm HBSS gently (to wash out media completely)

Add 1 ml TrypLE per Flask

Place flask in incubator (37° C.) for 1 min

Rock flask to detach cells

Add 9 ml D10+HEPES media (37° C.)

Pipette up and down 5 times to generate single cell suspension

Split at 1:2-1:2.5 (12 ml media for T75) ratio (if cells are growing more slowly, discard and thaw a new batch, they are not in optimal growth)

transfer to T225 as soon as enough cells are present (for ease of handling large amounts of cells)

AAV Production (Single 15 cm Dish Scale)

Plate 10 million cells in 21.5 ml media into a 15 cm dish

Incubate for 18-22 hours at 37° C.

Transfection is ideal at 80% confluence per plate

Prewarm 22 ml media (D10+HEPES)

Prepare tube with DNA mixture (use endofree maxiprep DNA):

-   -   5.2 ug vector of interest plasmid     -   8.7 ug AAV 1 serotype plasmid     -   10.4 ug DF6 plasmid (adenovirus helper genes)

Vortex to mix

Add 434 uL DMEM (no serum!)

Add 130 ul PEI solution

Vortex 5-10 seconds

Add DNA/DMEM/PEI mixture to prewarmed media

Vortex briefly to mix

Replace media in 15 cm dish with DNA/DMEM/PEI mixture

Return to 37° C. incubator

Incubate 48 h before harvesting (advantageously monitor to ensure medium is not turning too acidic)

Virus Harvest:

Remove supernatant from 15 cm dish

Filter with 0.45 um filter (low protein binding) Aliquot and freeze at −80° C.

Transduction (primary neuron cultures in 24-well format, 5DIV)

Replace complete neurobasal media in each well of neurons to be transduced with fresh neurobasal (usually 400 ul out of 500 ul per well is replaced)

Thaw AAV supernatant in 37° C. waterbath

Let equilibrate in incubator for 30 min

Add 250 ul AAV supernatant to each well

Incubate 24 h at 37° C.

Remove media/supernatant and replace with fresh complete neurobasal

Expression starts to be visible after 48 h, saturates around 6-7 Days Post Infection

Constructs for pAAV plasmid with GOI should not exceed 4.8 kb including both ITRS

AAV Supernatant Production

HEK 293FT cells (Life Technologies) were grown in antibiotic-free D10 media (DMEM high glucose with GlutaMax and Sodium Pyruvate, 10% heat-inactivated Hyclone FBS, and 1% 1M HEPES) and passaged daily at 1:2-2.5. The total number of passages was kept below 10 and cells were never grown beyond 85% confluence. The day before transfection, 1×10⁶ cells in 21.5 mL of D10 media were plated onto 15 cm dishes and incubated for 18-22 hours or until ˜80% confluence. For use as a transfection reagent, 1 mg/mL of PEI “Max” (Polysciences) was dissolved in water and the pH of the solution was adjusted to 7.1. For AAV production, 10.4 μg of pDF6 helper plasmid, 8.7 μg of pAAV1 serotype packaging vector, and 5.2 μg of pAAV vector carrying the gene of interest were added to 434 μL of serum-free DMEM and 1304, of PEI “Max” solution was added to the DMEM-diluted DNA mixture. The DNA/DMEM/PEI cocktail was vortexed and incubated at room temperature for 15 min. After incubation, the transfection mixture was added to 22 mL of complete media, vortexed briefly, and used to replace the media for a 15 cm dish of 293FT cells. For supernatant production, transfection supernatant was harvested at 48 hours, filtered through a 0.45 micron PVDF filter (Millipore), distributed into aliquots, and frozen for storage at −80° C.

To test the efficacy of AAV-mediated TALE delivery for modulating transcription in primary mouse cortical neurons, Applicants constructed six TALE-DNA binding domains targeting the genetic loci of three mouse neurotransmitter receptors: Grm5, Grin2a, and Grm2, which encode mGluR5, NMDA subunit 2A and mGluR2, respectively (FIG. 37C). To increase the likelihood of a target site accessibility, Applicants used mouse cortex DNase I sensitivity data from the UCSC genome browser to identify putative open chromatin regions. DNase I sensitive regions in the promoter of each target gene provided a guide for the selection of TALE binding sequences (FIG. 43). For each TALE, Applicants employed VP64 as a transcriptional activator or a quadruple tandem repeat of the mSin3 interaction domain (SID) (20, 30) as a repressor. Applicants have previously shown that a single SID fused to TALE downregulated a target gene effectively in 293FT cells (18). Hoping to further improve this TALE repressor, Applicants reasoned that four repeats of SID—analogous to the successful quadruple VP16 repeat architecture of VP64 (20)—might augment its repressive activity. This was indeed the case, as TALE-SID4X constructs enhanced repression ˜2-fold over TALE-SID in 293FT cells (FIG. 44).

Applicants found that four out of six TALE-VP64 constructs (T1, T2, T5 and T6) efficiently activated their target genes Grm5 and Grm2 in AAV-transduced primary neurons by up to 3- and 8-fold, respectively (FIG. 37C). Similarly, four out of six TALE-SID4X repressors (T9, T10, T11, T12) reduced the expression of their endogenous targets Grin2a and Grm2 by up to 2- and 8-fold, respectively (FIG. 37C). Together, these results indicate that constitutive TALEs can positively or negatively modulate endogenous target gene expression in neurons. Notably, efficient activation or repression by a given TALE did not predict its efficiency at transcriptional modulation in the opposite direction. Therefore, multiple TALEs may need to be screened to identify the most effective TALE for a particular locus.

As a confirmation of TALE expression and activity in vivo, Applicants performed stereotactic injection of concentrated AAV vectors into the mouse prefrontal cortex. Delivery of constitutive TALE-VP64 AAV vectors resulted in robust TALE expression in the mouse prefrontal cortex (FIG. 37D-E). Tissue punches from the AAV-transduced brain regions showed that a TALE-VP64 targeting the Grm2 gene locus is able to activate mRNA levels by up to 2.5-fold (FIG. 37F).

In order to deliver LITEs into neurons using AAV, Applicants had to ensure that the total viral genome size, with the LITE transgenes included, did not exceed 4.8 kb^(31,32). To that end, Applicants shortened the TALE N- and C-termini (keeping 136 aa in the N-terminus and 63 aa in the C-terminus) and exchanged the CRY2PHR and CIB1 domains (TALE-CIB1 and CRY2PHR-VP64; FIG. 38A). This switch allowed each component of LITE to fit into AAV vectors and did not reduce the efficacy of light-mediated transcription modulation (FIG. 45). These LITEs can be efficiently delivered into primary cortical neurons via co-transduction by a combination of two AAV vectors (FIG. 38B; delivery efficiencies of 83-92% for individual components with >80% co-transduction efficiency).

When implementing a neuron specific light-stimulation protocol, cultured neurons proved to be much more sensitive to blue light than Neuro-2a cells. Stimulation parameters that Applicants previously optimized for Neuro 2a cells (466 nm, 5 mW/cm² intensity, 7% duty cycle with 1 s light pulse at 0.067 Hz for a total of 24 h) caused >50% toxicity in primary neurons. Applicants therefore tested survival with a lower duty cycle, as Applicants had previously observed that a wide range of duty cycles had little effect on LITE-mediated transcriptional activation (FIG. 40).

For a neuronal application of LITEs, Applicants selected the Grm2 TALE (T6), which exhibited the strongest level of target upregulation in primary neurons, based on Applicants' comparison of 6 constitutive TALE activators (FIG. 37C). Applicants investigated its function using 2 light pulsing frequencies with the same duty cycle of 0.8%. Both stimulation conditions achieved a ˜7-fold light-dependent increase in Grm2 mRNA levels (FIG. 38C). Further study confirmed that, significant target gene expression increases could be attained quickly (4-fold upregulation within 4 h; FIG. 38D). In addition, Applicants observed significant upregulation of mGluR2 protein after stimulation, demonstrating that changes effected by LITEs at the mRNA level are translated to the protein domain (FIG. 38E). Taken together, these results confirm that LITEs enable temporally precise optical control of endogenous gene expression in neurons.

As a compliment to Applicants' previously implemented LITE activators, Applicants next engineered a LITE repressor based on the TALE-SID4X constructs. Constitutive Grm2 TALEs (T11 and T12, FIG. 38F) mediated the highest level of transcription repression, and were chosen as LITE repressors (FIG. 38F-G). Both light-induced repressors mediated significant downregulation of Grm2 expression, with 1.95-fold and 1.75-fold reductions for T11 and T12, respectively, demonstrating the feasibility of optically controlled repression in neurons (FIG. 38G).

Light-mediated control of gene expression would be particularly desirable in vivo. In contrast to current chemically inducible expression systems, LITEs have the potential for finer anatomical localization. Moreover, the kinetics of the system do not depend on drug diffusion, metabolism, or clearance, and stimulation can be achieved without drug-related side effects. To apply the LITE system in vivo, Applicants stereotactically delivered a 1:1 mixture of high concentration AAV vectors (10¹² DNAseI resistant particles/mL) carrying the Grm2-targeting T6-CIB1 and CRY2PHR-VP64 LITE components into the infralimbic cortex (ILC) of wildtype C57BL/6N mice. To provide optical stimulation of LITE-expressing neurons in vivo, Applicants also implanted a fiber optic cannula at the injection site (FIG. 38H)³³. Neurons in the injection site were efficiently co-transduced by both viruses, with >80% of transduced cells expressing both TALE12-CIB1 and CRY2PHR-VP64 (FIGS. 381 and 48). 8 days post-surgery, Applicants stimulated the ILC by connecting a solid-state 473 nm laser to the implanted fiber cannula. Following a 12 h stimulation period (5 mW, 0.8% duty cycle using 0.5 s light pulses at 0.0167 Hz), brain tissue from the fiber optic cannula implantation site was analyzed (FIG. 38H) for changes in Grm2 mRNA. Applicants observed a significant increase in Grm2 mRNA after light stimulation compared with unstimulated ILC (2.1-fold, p<0.01 vs. 1.3-fold background FIG. 38J), successfully demonstrating the utility of the LITE system for altering gene expression in vivo. This experiment suggests the potential value of LITEs for probing gene functions in the brain.

The investigation of dynamic transcriptional networks in heterogeneous tissues such as the brain would benefit greatly from spatiotemporally precise in vivo gene regulation. Such a system would allow researchers to ask questions about the role of dynamic gene regulation in processes as diverse as development, learning, memory, and disease progression. LITEs can be used to enable temporally precise, spatially-targeted, and bi-modal control of endogenous gene expression in cell lines, primary neurons, and in the mouse brain in vivo. The TALE DNA binding component of LITEs can be customized to target a wide range of genomic loci. Independently, novel functionalities can be achieved via alteration of the LITE effector domain. This system provides a powerful addition to existing optogenetic platforms, establishing a highly generalizable mode of altering endogenous gene transcription using light. Future work will increase the potency of LITE-mediated transcription modulation, reduce the level of background activity, and expand the range of wavelengths through which LITEs may be controlled. This may be achieved through exploration of other naturally occurring light-sensitive proteins³⁴⁻³⁷ or through directed evolution³⁸⁻⁴¹ of cryptochrome proteins. Finally, the modular design of the LITE system provides the opportunity for the development of a broad array of light-switchable tools for reverse-engineering genetic and epigenetic functions in a variety of biological systems.

LITE constructs were transfected into in Neuro 2A cells using GenJetAAV vectors carrying TALE or LITE constructs were used to transduce mouse primary embryonic cortical neurons as well as the mouse brain in vivo. RNA was extracted and reverse transcribed and mRNA levels were measured using TaqMan-based RT-qPCR. Light emitting diodes or solid-state lasers were used for light delivery in tissue culture and in vivo respectively.

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42. McClure, C., Cole, K. L., Wulff, P., Klugmann, M. & Murray, A. J. Production and titering of recombinant adeno-associated viral vectors. J Vis Exp, e3348 (2011).

Neuro 2a cells (Sigma-Aldrich) were grown in media containing a 1:1 ratio of OptiMEM (Life Technologies) to high-glucose DMEM with GlutaMax and Sodium Pyruvate (Life Technologies) supplemented with 5% HyClone heat-inactivated FBS (Thermo Scientific), 1% penicillin/streptomycin (Life Technologies), and passaged at 1:5 every 2 days. 120,000 cells were plated in each well of a 24-well plate 18-20 h prior to transfection. 1 h before transfection, media was changed to DMEM supplemented with 5% HyClone heat-inactivated FBS and 1% penicillin/streptomycin. Cells were transfected with 1.0 μg total of construct DNA (at equimolar ratios) per well with 1.5 μL of GenJet (SignaGen Laboratories) transfection reagent according to the manufacturer's instructions. Media was exchanged 24 h and 44 h post-transfection and light stimulation was started at 48 h. Stimulation parameters were: 5 mW/cm2, 466 nm, 7% duty cycle (1 s light pulse 0.067 Hz) for 24 h unless indicated otherwise in figure legends. RNA was extracted using the RNeasy kit (Qiagen) according to manufacturer's instructions and 1 μg of RNA per sample was reverse-transcribed using qScript (Quanta Biosystems). Relative mRNA levels were measured by quantitative real-time PCR (qRT-PCR) using TaqMan probes specific for the targeted gene as well as GAPDH as an endogenous control (Life Technologies, see Table 2 for Taqman probe IDs). ΔΔCt analysis was used to obtain fold-changes relative to negative controls transduced with GFP only and subjected to light stimulation. Toxicity experiments were conducted using the LIVE/DEAD assay kit (Life Technologies) according to instructions.

293FT cells (Life Technologies) were grown in antibiotic-free D10 media (DMEM high glucose with GlutaMax and Sodium Pyruvate, 10% heat-inactivated Hyclone FBS, and 1% 1M HEPES) and passaged daily at 1:2-2.5. The total number of passages was kept below 10 and cells were never grown beyond 85% confluence. The day before transfection, 1×10⁶ cells in 21.5 mL of D10 media were plated onto 15 cm dishes and incubated for 18-22 hours or until ˜80% confluence. For use as a transfection reagent, 1 mg/mL of PEI “Max” (Polysciences) was dissolved in water and the pH of the solution was adjusted to 7.1. For AAV production, 10.4 μg of pDF6 helper plasmid, 8.7 μg of pAAV1 serotype packaging vector, and 5.2 μg of pAAV vector carrying the gene of interest were added to 434 μL of serum-free DMEM and 130 μL of PEI “Max” solution was added to the DMEM-diluted DNA mixture. The DNA/DMEM/PEI cocktail was vortexed and incubated at room temperature for 15 min. After incubation, the transfection mixture was added to 22 mL of complete media, vortexed briefly, and used to replace the media for a 15 cm dish of 293FT cells. For supernatant production, transfection supernatant was harvested at 48 h, filtered through a 0.45 μm PVDF filter (Millipore), distributed into aliquots, and frozen for storage at −80° C.

Dissociated cortical neurons were prepared from C57BL/6N mouse embryos on E16 (Charles River Labs). Cortical tissue was dissected in ice-cold HBSS—(50 mL 10×HBSS, 435 mL dH₂O, 0.3 M HEPES pH 7.3, and 1% penicillin/streptomycin). Cortical tissue was washed 3× with 20 mL of ice-cold HBSS and then digested at 37° C. for 20 min in 8 mL of HBSS with 240 μL of 2.5% trypsin (Life Technologies). Cortices were then washed 3 times with 20 mL of warm HBSS containing 1 mL FBS. Cortices were gently triturated in 2 ml of HBSS and plated at 150,000 cells/well in poly-D-lysine coated 24-well plates (BD Biosciences). Neurons were maintained in Neurobasal media (Life Technologies), supplemented with 1×B27 (Life Technologies), GlutaMax (Life Technologies) and 1% penicillin/streptomycin.

Primary cortical neurons were transduced with 250 μL of AAV1 supernatant on DIV 5. The media and supernatant were replaced with regular complete neurobasal the following day. Neurobasal was exchanged with Minimal Essential Medium (Life Technologies) containing 1×B27, GlutaMax (Life Technologies) and 1% penicillin/streptomycin 6 days after AAV transduction to prevent formation of phototoxic products from HEPES and riboflavin contained in Neurobasal during light stimulation.

Light stimulation was started 6 days after AAV transduction (DIV 11) with an intensity of 5 mW/cm², duty cycle of 0.8% (250 ms pulses at 0.033 Hz or 500 ms pulses at 0.016 Hz), 466 nm blue light for 24 h unless indicated otherwise in figure legends. RNA extraction and reverse transcription were performed using the Cells-to-Ct kit according to the manufacturers instructions (Life Technologies). Relative mRNA levels were measured by quantitative real-time PCR (qRT-PCR) using TaqMan probes as described above for Neuro 2a cells.

For immunohistochemistry of primary neurons, cells were plated on poly-D-lysine/laminin coated coverslips (BD Biosciences) after harvesting. AAV1-transductions were performed as described above. Neurons were fixed 7 days post-transduction with 4% paraformaldehyde (Sigma Aldrich) for 15 min at RT. Blocking and permeabilization were performed with 10% normal goat serum (Life Technologies) and 0.5% Triton-X100 (Sigma-Aldrich) in DPBS (Life Technologies) for 1 h at room temperature. Neurons were incubated with primary antibodies overnight at 4° C., washed 3× with DPBS and incubated with secondary antibodies for 90 min at RT. For antibody providers and concentrations used, see Table 3. Coverslips were finally mounted using Prolong Gold Antifade Reagent with DAPI (Life Technologies) and imaged on an Axio Scope A.1 (Zeiss) with an X-Cite 120Q light source (Lumen Dynamics). Image were acquired using an AxioCam MRm camera and AxioVision 4.8.2.

For preparation of total protein lysates, primary cortical neurons were harvested after light stimulation (see above) in ice-cold lysis buffer (RIPA, Cell Signaling; 0.1% SDS, Sigma-Aldrich; and cOmplete ultra protease inhibitor mix, Roche Applied Science). Cell lysates were sonicated for 5 min at ‘M’ setting in a Bioruptor sonicator (Diagenode) and centrifuged at 21,000×g for 10 min at 4° C. Protein concentration was determined using the RC DC protein assay (Bio-Rad). 30-40 μg of total protein per lane was separated under non-reducing conditions on 4-15% Tris-HCl gels (Bio-Rad) along with Precision Plus Protein Dual Color Standard (Bio-Rad) After wet electrotransfer to polyvinylidene difluoride membranes (Millipore) and membrane blocking for 45 min in 5% BLOT-QuickBlocker (Millipore) in Tris-buffered saline (TBS, Bio-Rad), western blots were probed with anti-mGluR2 (Abcam, 1:1.000) and anti-α-tubulin (Sigma-Aldrich 1:20,000) overnight at 4° C., followed by washing and anti-mouse-IgG HRP antibody incubation (Sigma-Aldrich, 1:5,000-1:10,000). For further antibody details see Table 3. Detection was performed via ECL Western blot substrate (SuperSignal West Femto Kit, Thermo Scientific). Blots were imaged with an AlphaImager (Innotech) system, and quantified using ImageJ software 1.46r.

Production of concentrated and purified AAV for stereotactic injection in-vivo was done using the same initial steps outlined above for production of AAV1 supernatant. However, for transfection, equal ratios of AAV1 and AAV2 serotype plasmids were used instead of AAV1 alone. 5 plates were transfected per construct and cells were harvested with a cell-scraper 48 h post transfection. Purification of AAV1/2 particles was performed using HiTrap heparin affinity columns (GE Healthcare)⁴². Applicants added a second concentration step down to a final volume of 100 μl per construct using an Amicon 500 μl concentration column (100 kDa cutoff, Millipore) to achieve higher viral titers. Titration of AAV was performed by qRT-PCR using a custom Taqman probe for WPRE (Life Technologies). Prior to qRT-PCR, concentrated AAV was treated with DNaseI (New England Biolabs) to achieve a measurement of DNaseI-resistant particles only. Following DNaseI heat-inactivation, the viral envelope was degraded by proteinase K digestion (New England Biolabs). Viral titer was calculated based on a standard curve with known WPRE copy numbers.

Adult (10-14 weeks old) male C57BL/6N mice were anaesthetized by intraperitoneal (i.p.) injection of Ketamine/Xylazine (100 mg/kg Ketamine and 10 mg/kg Xylazine) and pre-emptive analgesia was given (Buprenex, 1 mg/kg, i.p.). Craniotomy was performed according to approved procedures and 1 μl of AAV1/2 was injected into ILC at 0.35/1.94/−2.94 (lateral, anterior and inferior coordinates in mm relative to bregma). During the same surgical procedure, an optical cannula with fiber (Doric Lenses) was implanted into ILC unilaterally with the end of the optical fiber located at 0.35/1.94/−2.64 relative to bregma. The cannula was affixed to the skull using Metabond dental cement (Parkell Inc) and Jet denture repair (Lang dental) to build a stable cone around it. The incision was sutured and proper post-operative analgesics were administered for three days following surgery.

Mice were injected with a lethal dose of Ketamine/Xylazine anaesthetic and transcardially perfused with PBS and 4% paraformaldehyde (PFA). Brains were additionally fixed in 4% PFA at 4° C. overnight and then transferred to 30% sucrose for cryoprotection overnight at room temperature. Brains were then transferred into Tissue-Tek Optimal Cutting Temperature (OCT) Compound (Sakura Finetek) and frozen at −80° C. 18 μm sections were cut on a cryostat (Leica Biosystems) and mounted on Superfrost Plus glass slides (Thermo Fischer). Sections were post-fixed with 4% PFA for 15 min, and immunohistochemistry was performed as described for primary neurons above.

8 days post-surgery, awake and freely moving mice were stimulated using a 473 nm laser source (OEM Laser Systems) connected to the optical implant via fiber patch cables and a rotary joint. Stimulation parameters were the same as used on primary neurons: 5 mW (total output), 0.8% duty cycle (500 ms light pulses at 0.016 Hz) for a total of 12 h. Experimental conditions, including transduced constructs and light stimulation are listed in Table 4.

After the end of light stimulations, mice were euthanized using CO2 and the prefrontal cortices (PFC) were quickly dissected on ice and incubated in RNA later (Qiagen) at 4° C. overnight. 200 μm sections were cut in RNA later at 4° C. on a vibratome (Leica Biosystems). Sections were then frozen on a glass coverslide on dry ice and virally transduced ILC was identified under a fluorescent stereomicroscope (Leica M165 FC). A 0.35 mm diameter punch of ILC, located directly ventrally to the termination of the optical fiber tract, was extracted (Harris uni-core, Ted Pella). The brain punch sample was then homogenized using an RNase-free pellet-pestle grinder (Kimble Chase) in 50 μl Cells-to-Ct RNA lysis buffer and RNA extraction, reverse transcription and qRT-PCR was performed as described for primary neuron samples.

All experiments were performed with a minimum of three independent biological replicates. Statistical analysis was performed with Prism (GraphPad) using student's t-test when comparing two conditions, ANOVA with Tukey's post-hoc analysis when comparing multiple samples with each other, ANOVA with Duncan's post-hoc analysis when comparing multiple samples to the negative control, and two-way ANOVA with Bonferroni post-hoc analysis to compare multiple groups over time.

Example 7 Development of AAV1 Supernatant Process

Traditional AAV particle generation required laborious production and purification processes, and made testing many constructs in parallel impractical (4). In this study, a simple yet highly effective process of AAV production using filtered supernatant from transfected 293FT cells (FIG. 42). Recent reports indicate that AAV particles produced in 293FT cells could be found not only it the cytoplasm but also at considerable amounts in the culture media (5). The ratio of viral particles between the supernatant and cytosol of host cells varied depending on the AAV serotype, and secretion was enhanced if polyethylenimine (PEI) was used to transfect the viral packaging plasmids (5). In the current study, it was found that 2×10⁵ 293 FT cells transfected with AAV vectors carrying TALEs (FIG. 37A) and packaged using AAV1 serotype were capable of producing 250 μl of AAV1 at a concentration of 5.6±0.24×10¹⁰ DNAseI resistant genome copies (gc) per mL. 250 μl of filtered supernatant was able to transduce 150,000 primary cortical neurons at efficiencies of 80-90% (FIGS. 37B and 42B). This is a dramatic increase over the 1-2% transduction efficiency achieved using lentivirus supernatant produced from the same number of 293FT cells (FIG. 42B).

TABLE 2 Product information for all Taqman probes (Life Technologies) Target Species Probe # Ngn2 mouse Mm00437603_g1 Grm5 (mGluR5) mouse Mm00690332_m1 Grm2 (mGluR2) mouse Mm01235831_m1 Grin2a (NMDAR2A) mouse Mm00433802_m1 GAPD (GAPDH) mouse 4352932E KLF4 human Hs00358836_m1 GAPD (GAPDH) human 4352934E WPRE custom

TABLE 3 Clone, product numbers and concentrations for antibodies used in this study Primary Antibodies Target Host Clone # Manufacturer Product # IsoType Concentration mGluR2 mouse mG2Na-s Abcam Ab15672 IgG 1:1000 α-tubulin mouse B-5-1-2 Sigma-Aldrich T5168 IgG1 1:20000 NeuN mouse A60 Millipore MAB377 IgG1 1:200 HA (Alexa Fluor mouse 6E2 Cell Signaling 3444 IgG1 1:100 594 conjugated) GFP chicken polyclonal Aves Labs GFP-1020 IgY 1:500 Secondary Antibodies Target Host Conjugate Manufacturer Product # Concentration mouse IgG goat HRP Sigma-Aldrich A9917 1:5000-10000 mouse IgG goat Alexa Fluor 594 Life Technologies A11005 1:1000 chicken IgG Goat Alexa Fluor 488 Life Technologies A11039 1:1000

TABLE 4 Viral transduction and light stimulation parameters for in vivo LITE-mediated activation of Grm2 in the mouse infralimbic cortex (ILC). Grm2 mRNA levels in the ipsilateral LITE-expressing hemisphere are compared with the contralateral mCherry-expressing control hemisphere for all three experimental conditions shown in FIG. 38J. ILC ILC Hemisphere (ipsilateral) Hemisphere Experimental Light (contralateral) condition AAV vector stimulation AAV vector GFP GFP yes mCherry LITEs/ TALE-CIB1::CRY2PHR- no mCherry no Light VP64 LITEs/ TALE-CIB1::CRY2PHR- yes mCherry +Light VP64

Sequences of constructs used in Neuro-2A cells (FIGS. 35, 36)

>TALE(Ngn2) (underlined)-NLS (in italics)-CRY2 (in bold)

(SEQ ID NO: 168) MSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLR AADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTV AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA IASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLC QAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETV QRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLG GRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQF GMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFA DSLERDLDAPSPMHEGDQTRAS ASPKKKRKVEAS KMDKKTIVWFRRDLRIEDNPALAAAAHEGS VFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGA TKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCL DMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNE FIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLF LRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWAT GWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDN PALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARE LLAKAISRTREAQIMIGAAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRV KPEEEEERDMKKSRGFDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLG KNG 

>TALE(Ngn2) (underlined)-NLS (in italics)-CRY2PHR (in bold)

(SEQ ID NO: 169) MSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLR AADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTV AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH DGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA IASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPE QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHG LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLC QAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETV QRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLG GRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQF GMSRHGLLQLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFA DSLERDLDAPSPMHEGDQTRAS ASPKKKRKVEAS KMDKKTIVWFRRDLRIEDNPALAAAAHEGS VFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGA TKVVFNHLYDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCL DMSIESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNE FIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLF LRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWAT GWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDN PALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARE LLAKAISRTREAQIMIGAAP 

>CIB1 (in bold)-NLS (in italics)-VP64 (in bold, underlined) _(—)2A_ GFP (underlined)

(SEQ ID NO: 170) MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRRE KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAK EVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPS MWDSHVQNLYGNLGV ASPKKKRKVEAS GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDMLGSDALDDFDLDMLIN SRGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGD VNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFK SAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNE KRDHMVLLEFVTAAGITLGMDELYK 

>CIBN (in bold)-NLS (in italics)-VP64 (in bold, underlined) _(—)2A_ GFP (underlined)

(SEQ ID NO: 171) MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYI ASPKKKRKVEAS GSGRADALDD FDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN SRGSGEGRGSLLTCGDV EENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNT PIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK 

>CIB1 (in bold)-NLS (in italics)-VP16 (in bold, underlined) _(—)2A_ GFP (underlined)

(SEQ ID NO: 172) MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRRE KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAK EVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPS MWDSHVQNLYGNLGV ASPKKKRKVEAS APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGD GDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGEFPGIRR SRGSGEGRGSLLTCGD VEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK 

>CIB1 (in bold)-NLS (in italics)-p65 (in bold, underlined) _(—)2A_ GFP (underlined)

(SEQ ID NO: 173) MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLP MMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKI TEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHSIAERVRRE KISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDFDMDDIFAK EVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSCFNNGEAPS MWDSHVQNLYGNLGV ASPKKKRKVEAS PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPP APAPVLTPGPPQSLSAPVPHSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVD NSEFQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDEDFS SIADMDFSALLSQISSSGQ SRGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKS AMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNV YIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEK RDHMVLLEFVTAAGITLGMDELYK 

AAV constructs (constructs used in primary neurons and in-vivo, FIGS. 37-38)

>HA-TALE(12mer) (in bold)-NLS (in italics)-VP64 (in bold, underlined) _(—)2A_ GFP (underlined)

(SEQ ID NO: 174) MYPYDVPDYAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVT AVEAVHAWRNALTGAPLNLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASX XGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVA IASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPE QVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLC QAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLL PVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGRPALESI VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA ASP KKKRKVEAS GSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML IN SRGSGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATY GKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKI RHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITL GMDELYK 

>HA-TALE(12mer) (in bold)-NLS (in italics)-SID4X (in bold, underlined) _(—)2A_ phiLOV2.1 (underlined)

(SEQ ID NO: 175) MYPYDVPDYAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVT AVEAVHAWRNALTGAPLNLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASX XGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVA IASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPE QVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLC QAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLL PVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGRPALESI VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA ASP KKKRKVEASPKKKRKVEAS GSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAA DYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAA DYLERREREAEHGYASMLPSR SRGSGEGRGSLLTCGDVEENPGPIEKSFVITDPRLPDYPIIFA SDGFLELTEYSREEIMGRNARFLQGPETDQATVQKIRDAIRDQRETTVQLINYTKSGKKFWNLL HLQPVRDRKGGLQYFIGVQLVGSDHV 

>HA-TALE(12mer) (in bold)-NLS (in italics)-CIB1 (underlined)

(SEQ ID NO: 176) MYPYDVPDYAVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTV AVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVT AVEAVHAWRNALTGAPLNLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASX XGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVA IASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPE QVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLC QAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGKQALETVQRLL PVLCQAHGLTPEQVVAIASXXGGKQALETVQRLLPVLCQAHGLTPEQVVAIASXXGGRPALESI VAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA ASP KKKRKVEAS NGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYL STAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEE GEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIHVRARRGQATDSHS IAERVRREKISERMKFLQDLVPGCDKITGKAGMLDEIINYVQSLQRQIEFLSMKLAIVNPRPDF DMDDIFAKEVASTPMTVVPSPEMVLSGYSHEMVHSGYSSEMVNSGYLHVNPMQQVNTSSDPLSC FNNGEAPSMWDSHVQNLYGNLGV 

>CRY2PHR(in bold)-NLS (in italics)-VP64 (in bold, underlined) _(—)2A_ GFP (underlined)

(SEQ ID NO: 177) MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLS QSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQS YNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEEL GLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEI SVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFP WDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDT LLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHP WDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAP ASPKKKRKVEAS G S G RADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLIN SRGSGEGR GSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQ LADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKV

>CRY2PHR (in bold)-NLS (in italics)-SID4X (in bold, underlined) _(—)2A_ phiLOV2.1 (underlined)

(SEQ ID NO: 178) MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSLAHLS QSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGISVQS YNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWACSIEEL GLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEI SVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFP WDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDT LLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHP WDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAAP ASPKKKRKVEAS G SGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPG SGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPS R SRGSGEGRGSLLTCGDVEENPGPIEKSFVITDPRLPDYPIIFASDGFLELTEYSREEIMGRNA RFLQGPETDQATVQKIRDAIRDQRETTVQLINYTKSGKKFWNLLHLQPVRDRKGGLQYFIGVQL VGSDHV 

Sequences of FIGS. 39-48

>TALE(KLF4) (underlined)-NLS (in italics)-CRY2PHR (in bold)

(SEQ ID NO: 179) MSRTRLPSPPAPSPAFSADSFSDLLRQFDPSLFNTSLFDSLPPFGAHHTEAATGEWDEVQSGLR AADAPPPTMRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGYSQQQQEKIKPKVRSTV AQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNGGGK QALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASN GGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVA IASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLC QAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAV KKGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLL QLFRRVGVTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLD APSPMHEGDQTRAS ASPKKKRKVEAS KMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWC PEEEGQFYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHL YDPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVM LPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLID YAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLRE YSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIR VIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKY DPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISR TREAQIMIGAAP_

>HA-NLS (in italics)-TALE(p11, N136) (in bold)-SID (underlined)

(SEQ ID NO: 180) MYPYDVPDYASPKKKRKVEASVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQ LLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGL TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH GLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHA PALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVG VTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHE GDQTRASAS GSGMNIQMLLEAADYLERREREAEHGYASMLP. 

>HA-NLS (in italics)-TALE(p11, N136) (in bold)-SID4X (underlined)

(SEQ ID NO: 181) MYPYDVPDYASPKKKRKVEASVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVA LSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQ LLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGL TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLP VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH GLTPEQVVAIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHA PALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSRHGLLQLFRRVG VTELEARSGTLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFADSLERDLDAPSPMHE GDQTRASAS GSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREA EHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREA EHGYASMLPSR 

The following Arduino script was used to enable the individual control of each 4-well column of a light-stimulated 24-well plate:

//Basic control code for LITE LED array using Arduino UNO //LED column address initialization to PWM-ready Arduino outputs int led1_pin = 3; int led2_pin = 5; int led3_pin = 6; int led4_pin = 9; int led5_pin = 10; int led6_pin = 11; //Maximum setting for Arduino PWM int uniform_brightness = 255; //PWM settings for individual LED columns int led1_brightness = uniform_brightness/2; int led2_brightness = uniform_brightness/2; int led3_brightness = uniform_brightness/2; int led4_brightness = uniform_brightness/2; int led5_brightness = uniform_brightness/2; int led6_brightness = uniform_brightness/2; //‘on’ time in msec unsigned long uniform_stim_time = 1000; / //individual ‘on’ time settings for LED columns unsigned long_led1_stim_time = uniform_stim_time; unsigned long_led2_stim_time = uniform_stim_time; unsigned long_led3_stim_time = uniform_stim_time; unsigned long_led4_stim_time = uniform_stim_time; unsigned long_led5_stim_time = uniform_stim_time; unsigned long_led6_stim_time = uniform_stim_time; //‘off’ time in msec unsigned long uniform_off_time = 14000; //individual ‘off’ time settings for LED columns unsigned long led1_off_time = uniform_off_time; unsigned long led2_off_time = uniform_off_time; unsigned long led3_off_time = uniform_off_time; unsigned long led4_off_time = uniform_off_time; unsigned long led5_off_time = uniform_off_time; unsigned long led6_off_time = uniform_off_time; unsigned long currentMillis = 0; //initialize timing and state variables unsigned long led1_last_change = 0; unsigned long led2_last_change = 0; unsigned long led3_last_change = 0; unsigned long led4_last_change = 0; unsigned long led5_last_change = 0; unsigned long led6_last_change = 0; int led1_state = HIGH; int led2_state = HIGH; int led3_state = HIGH; int led4_state = HIGH; int led5_state = HIGH; int led6_state = HIGH; unsigned long led1_timer = 0; unsigned long led2_timer = 0; unsigned long led3_timer = 0; unsigned long led4_timer = 0; unsigned long led5_timer = 0; unsigned long led6_timer = 0; void setup( ) { // setup PWM pins for output pinMode(led1_pin, OUTPUT); pinMode(led2_pin, OUTPUT); pinMode(led3_pin, OUTPUT); pinMode(led4_pin, OUTPUT); pinMode(led5_pin, OUTPUT); pinMode(led6_pin, OUTPUT); //LED starting state analogWrite(led1_pin, led1_brightness); analogWrite(led2_pin, led2_brightness); analogWrite(led3_pin, led3_brightness); analogWrite(led4_pin, led4_brightness); analogWrite(led5_pin, led5_brightness); analogWrite(led6_pin, led6_brightness); } void loop( ) { currentMillis = millis( ); //identical timing loops for the 6 PWM output pins led1_timer = currentMillis − led1_last_change; if (led1_state == HIGH){ //led state is on if (led1_timer >= led1_stim_time){ //TRUE if stim time is complete analogWrite(led1_pin, 0); //turn LED off led1_state = LOW; //change LED state variable led1_last_change = currentMillis; //mark time of most recent change } } else{ //led1 state is off if (led1_timer >= led1_off_time){ //TRUE if off time is complete analogWrite(led1_pin, led1_brightness); //turn LED on led1_state = HIGH; //change LED state variable led1_last_change = currentMillis; //mark time of most recent change } } led2_timer = currentMillis − led2_last_change; if (led2_state == HIGH){ if (led2_timer >= led2_stim_time){ analogWrite(led2_pin, 0); led2_state = LOW; led2_last_change = currentMillis; } } else{ //led2 state is off if (led2_timer >= led2_off_time){ analogWrite(led2_pin, led2_brightness); led2_state = HIGH; led2_last_change = currentMillis; } } led3_timer = currentMillis − led3_last_change; if (led3_state == HIGH){ if (led3_timer >= led3_stim_time){ analogWrite(led3_pin, 0); led3_state = LOW; led3_last_change = currentMillis; } } else{ //led3 state is off if (led3_timer >= led3_off_time){ analogWrite(led3_pin, led3_brightness); led3_state = HIGH; led3_last_change = currentMillis; } } led4_timer = currentMillis − led4_last_change; if (led4_state == HIGH){ if (led4_timer >= led4_stim_time){ analogWrite(led4_pin, 0); led4_state = LOW; led4_last_change = currentMillis; } } else{ //led4 state is off if (led4_timer >= led4_off_time){ analogWrite(led4_pin, led4_brightness); led4_state = HIGH; led4_last_change = currentMillis; } } led5_timer = currentMillis − led5_last_change; if (led5_state == HIGH){ if (led5_timer >= led5_stim_time){ analogWrite(led5_pin, 0); led5_state = LOW; led5_last_change = currentMillis; } } else{ //led5 state is off if (led5_timer >= led5_off_time){ analogWrite(led5_pin, led5_brightness); led5_state = HIGH; led5_last_change = currentMillis; } } led6_timer = currentMillis − led6_last_change; if (led6_state == HIGH){ if (led6_timer >= led6_stim_time){ analogWrite(led6_pin, 0); led6_state = LOW; led6_last_change = currentMillis; } } else{ //led6 state is off if (led6_timer >= led6_off_time){ analogWrite(led6_pin, led6_brightness); led6_state = HIGH; led6_last_change = currentMillis; } } }

REFERENCES

-   1 Banerjee, R. et al. The Signaling State of Arabidopsis     Cryptochrome 2 Contains Flavin Semiquinone. Journal of Biological     Chemistry 282, 14916-14922, doi:10.1074/jbc.M700616200 (2007). -   2 McClure, C., Cole, K. L., Wulff, P., Klugmann, M. & Murray, A. J.     Production and titering of recombinant adeno-associated viral     vectors. J Vis Exp, e3348, doi:10.3791/3348 (2011). -   3 Witten, Ilana B. et al. Recombinase-Driver Rat Lines: Tools,     Techniques, and Optogenetic Application to Dopamine-Mediated     Reinforcement. Neuron 72, 721-733, doi: at the website     dx.doi.org/10.1016/j.neuron.2011.10.028 (2011). -   4 Grieger, J. C., Choi, V. W. & Samulski, R. J. Production and     characterization of adeno-associated viral vectors. Nat Protoc 1,     1412-1428, doi:10.1038/nprot.2006.207 (2006). -   5 Lock M, A. M., Vandenberghe L H, Samanta A, Toelen J, Debyser Z,     Wilson J M. Rapid, Simple, and Versatile Manufacturing of     Recombinant Adeno-Associated Viral Vectors at Scale. Human Gene     Therapy 21, 1259-1271, doi:10.1089/hum.2010.055 (2010).

Example 8 Cloning (Construction) of AAV Constructs

Construction of AAV-Promoter-TALE-Effector Backbone

For construction of AAV-promoter-TALE-effector a backbone was cloned by standard subcloning methods. Specifically, the vector contained an antibiotics resistance gene, such as ampicillin resistance and two AAV inverted terminal repeats (itr's) flanking the promoter-TALE-effector insert (sequences, see below). The promoter (hSyn), the effector domain (VP64, SID4X or CIB1 in this example)/the N- and C-terminal portion of the TALE gene containing a spacer with two typeIIS restriction sites (BsaI in this instance) were subcloned into this vector. To achieve subcloning, each DNA component was amplified using polymerase-chain reaction and then digested with specific restriction enzymes to create matching DNA sticky ends. The vector was similarly digested with DNA restriction enzymes. All DNA fragments were subsequently allowed to anneal at matching ends and fused together using a ligase enzyme.

Assembly of Individual TALEs into AAV-Promoter-TALE-Effector Backbone

For incorporating different TALE monomer sequences into the AAV-promoter-TALE-effector backbone described above, a strategy based on restriction of individual monomers with type IIS restriction enzymes and ligation of their unique overhangs to form an assembly of 12 to 16 monomers to form the final TALE and ligate it into the AAV-promoter-TALE-effector backbone by using the type IIS sites present in the spacer between the N- and C-term (termed golden gate assembly). This method of TALE monomer assembly has previously been described by us (NE Sanjana, L Cong, Y Zhou, M M Cunniff, G Feng & F Zhang A transcription activator-like effector toolbox for genome engineering Nature Protocols 7, 171-192 (2012) doi:10.1038/nprot.2011.431)

By using the general cloning strategy outlined above, AAV vectors containing different promoters, effector domains and TALE monomer sequences can be easily constructed.

Nucleotide Sequences:

Left AAV ITR (SEQ ID NO: 182) Cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggc gacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatc actaggggttcct_ Right AAV ITR (SEQ ID NO: 183) Aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgg gcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgc agctgcctgcagg_ hSyn promoter (SEQ ID NO: 184) gtgtctagactgcagagggccctgcgtatgagtgcaagtgggttttaggaccaggatgaggcgg ggtgggggtgcctacctgacgaccgaccccgacccactggacaagcacccaacccccattcccc aaattgcgcatcccctatcagagagggggaggggaaacaggatgcggcgaggcgcgtgcgcact gccagcttcagcaccgcggacagtgccttcgcccccgcctggcggcgcgcgccaccgccgcctc agcactgaaggcgcgctgacgtcactcgccggtcccccgcaaactccccttcccggccaccttg gtcgcgtccgcgccgccgccggcccagccggaccgcaccacgcgaggcgcgagataggggggca cgggcgcgaccatctgcgctgcggcgccggcgactcagcgctgcctcagtctgcggtgggcagc ggaggagtcgtgtcgtgcctgagagcgcagtcgagaa_ TALE N-term (+136 AA truncation) (SEQ ID NO: 185) GTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGT CGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGC CTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCG TTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGC TTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCA GTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAAT GCGCTCACGGGAGCACCCCTCAAC_ TALE C-term (+63 AA truncation) (SEQ ID NO: 186) CGGACCCCGCGCTGGCCGCACTCACTAATGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACG ACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAAC AGAAGGATTCCCGAGAGGACATCACATCGAGTGGCA_ Ampicillin resistance gene (SEQ ID NO: 187) atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttt ttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtggg ttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgtttt ccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggc aagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcac agaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagt gataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttt tgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccat accaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactatta actggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaag ttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagc cggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatc gtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgaga taggtgcctcactgattaagcattgg_

Example 9 DNA Ratios

In this application, Applicants provide for varying plasmid ratios. The ratios of vector of interest plasmid: AAV serotype plasmid: pHelper plasmid may be varied. Specific values used in examples above are: 1:1.7:2 for AAV supernatant production down to 24-well scale. Values that may be used for production in 96-well format are: 1:2:1. Values may be varied in a wider range (e.g. up to fivefold excess of one plasmid) if desired.

Scalability

The present invention also comprehends AAV supernatant production as described herein being easily scaled up into higher throughput formats. The examples listed describe scaling from 15 cm dishes to 96-well plates for production. Through the same principle of scaling it may be possible to produce AAV in more dense well plate formats (e.g. 384-well, 1536-well etc.). The invention further comprehends using this process in even smaller volume units as would be possible with e.g. a microfluidic device capable of maintaining cell cultures in individual chambers. Hence, the present invention allows for an unprecedented throughput of production of different AAV viral particles. Applicants submit that one further important advantage of the invention described is that due to the highly efficient recovery of functional viral particles (due to minimal loss compared to extensive purification procedures traditionally used) AAV supernatant can be produced at the same scale as it will be applied. This is especially relevant for automated processing as it provides not only a simplified production and application process but also reduces the possibility for variability. In a preferred embodiment, the invention comprehends the automated production of 96 different AAV particles in 96-well plate format and application of the harvested supernatant to 3 replicate plates of cells to be transduced. This requires minimal pipetting steps, no necessary rearrangement (entire plates of virus can be applied to cells with a 96-channel pipette head) and minimal chance of pipetting error.

Filtering/Purification

Multiple methods may be used to purify the cell supernatant containing AAV particles after harvest and before application to cells for transduction. For a basic purification which mostly serves to remove any potential 293FT cells and large cell debris from the supernatant, filtration with a 22 micron or 45 micron pore size low protein binding filter or centrifugation for pelleting cells and cell debris may be employed. In the case of filtration, the flow-through will be harvested and used subsequently and in the case of centrifugation (at speeds in a range of e.g. 200 g for 10 min to 6000 g for 1-10 min) the supernatant will be used. In cases where more stringent purification is desired (e.g. for particularly sensitive cell types such as human ES cells or in a clinical application) it may be possible to follow up with subsequent purification steps. In an aspect of the invention, a sequence of molecular weight cutoff filters may be used (e.g. Amicon filters, millipore).

FBS Substitutes

The use of fetal bovine serum in the production of supernatant AA V may prove problematic for certain downstream applications. For example, the application of FBS-containing AAV supernatant to embryonic stem cells would result in uncontrolled differentiation of the pluripotent cultures. Also, the use of undefined FBS is incompatible with human clinical applications. In order to mitigate the issues arising from the use of FBS, the invention comprehends the culture medium used to support the AAV producing 293FT cells being replaced with a chemically-defined serum-free medium. For example, Pro293a from Lanza Biologics is a chemically-defined, serum-free medium designed to support the growth and protein production of adherent 293 lineage cells. With regards to the AAV supernatant production protocol details in the examples herein, all media components would simply be replaced with Pro293a or another suitable medium substitute.

Reasons to Use AAV

Non-integration: A major motivation for the use of AAV in the field of gene therapy is the relative lack of random genomic integration compared to lentivirus, retrovirus, and other integrating viral vectors. The majority of transduced recombinant AAV genetic material exists in the host cell as episomes, rather than at randomly integrated chromosomal locations. In human cells, if the appropriate helper genes are provided, the AAV genome can integrate at the well-characterized safe harbor locus AAVS 1. These characteristics reduce the chance for oncogenic integration, making AAV the current preferred viral system for human gene therapy. The non-integration of AAV also provides advantages for functional genomic studies. By providing trans genes or expression modulation systems via AAV, rather than an integrating virus, one can be assured that the cell population being used maintains an otherwise isogenic background.

Functional Genomics: Cell Type Addressability

The generation of large libraries of RNAi, ORFs, targeted nucleases (ZFNs, TALENs, CRISPR/Cas9), transcriptional modulators (TALE-TFs, CRISPR/dCAS9 effectors), and other gene expression tools has enabled large-scale arrayed functional genomics. These types of experiments, however, are limited to cell types to which such gene expression tools can be delivered in high-throughput. The high-throughput scalability of Applicants' AAV supernatant production protocol allows for the application of functional genomics techniques to cell types for which AAV is the ideal delivery mechanism. For example, AAV may be used to transduce primary cortical neurons with higher efficiency than lentiviral transduction or plasmid transfection, with lower toxicity than lentiviral delivery.

Pooling

The herein described AAV supernatant production method may be used to generate functional, pooled AAV supernatant. In an embodiment of the invention, several genes of interest, encoded on separate AAV backbone plasmids can be pooled at the plasmid stage to produce a final supernatant containing a mixture of the desired AAV vectors. Several types of gene delivery applications may benefit from a pooling approach. First, some experiments in which a large number of viral vectors must be functionally tested could be performed in a hierarchical pooled fashion. For example, groups of multiple RNAi or ORFs could be delivered in pooled AAV format to reduce the size of the initial search space, saving experimental time and cost. Second, complicated multicomponent gene expression systems may be produced via a pooled AAV format. For example, the differentiation of embryonic stem cells or reprogramming of one cell type to another often requires the delivery of numerous transcription factors simultaneously. Methods of the invention encompassing pooled AAV supernatant production could rapidly provide many different transcription factor combinations, simply by altering the mixtures of AAV backbone plasmids, which may be automated by liquid handling robotics. Third, artificial transcription factors, such as TALE-TFs and CRISPR/Cas9 activators, have been shown to have synergistic effects when provided in combination to target cells. Pooled AAV supernatant production could rapidly provide many different TALE-TF, CRISPR/Cas9, or other engineered gene expression modulators, simply by altering the mixtures of AAV backbone plasmids. This approach has been validated for pooled TALE-TFs designed to activate gene expression in mouse primary cortical neurons. Ten separate TALE-VP64 activators designed to target the Drd2 locus were produced by Applicants' standard AAV supernatant production method. Simultaneously, an equimolar mixture of all10 Drd2 targeting TALE-VP64 plasmids was made, referred to as the “10 TALE mixture”. The identical AAV supernatant production protocol was used produce the pooled AAV mixture, with the exception that the gene of interest backbone plasmid was replaced by an equal mass of “10 TALE mixture” plasmids. All AAV supernatants were harvested and applied to mouse primary neuron cultures as previously described. Six days after transduction, cell lysis, reverse transcription and qPCR were performed on the neuron cultures to determine the expression levels of Drd2. Gene expression levels were elevated for several of the TALE-VP64 transduced cultures. The culture transduced with supernatant from the “10 TALE mixture” was found to activate expression from the Drd2 locus at a level equivalent to the most potent individual TALE-VP64.

Multiple Harvests

Multiple supernatant AAV batches may be harvested from a single AAV producing 293FT culture. Specifically, following the 48 hour post-transfection harvested described in Applicants' standard AAV supernatant protocol, the culture medium may be replenished and harvested again 24 hours later (72 hours post-transfection). Both harvests contain functional AAV particles. In this presently described multiple harvest protocol, the value of producing twice as much AAV supernatant as Applicants' standard protocol saves time and resources when producing many AAV cultures in an arrayed format. This approach offers an advantage over current large-scale AAV production methods. In current methods, the amount of AAV that can be produced is limited by the mass of 293 cells producing the viral particles, as these methods typically require lysing the producer cells to harvest the AAV particles. By stably expressing the AAV expression plasmids in a 293 producer cell line, one could continually harvest AAV supernatant batches simply by maintaining the cell cultures, periodically collecting the supernatant, and replenishing the culture medium.

In additional embodiments, the invention comprises a method for obtaining and optionally storing a sample containing a set amount of a Dependovirus-based vector comprising or consisting essentially of: (a) creating infected or transfected cells by a process comprising or consisting essentially of one or more methods selected from: (i) transfecting plasmid(s) containing or consisting essentially of exogenous DNA including DNA for expression into Dependovirus-based vector-infected cells along with another helper plasmid that provides Dependovirus rep and/or cap genes which are obligatory for replication and packaging of the Dependovirus-based vector; or (ii) infecting susceptible cells with a Dependovirus-based vector containing or consisting essentially of exogenous DNA including DNA for expression, and helper virus wherein the Dependovirus-based vector lacks functioning cap and/or rep and the helper virus provides the cap and/or rev function that the Dependovirus-based vector lacks; or (iii) infecting susceptible cells with a Dependovirus-based vector containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, and transfecting said cells with a plasmid supplying cap and/or rep function that the Dependovirus-based vector lacks; or (iv) infecting susceptible cells with a Dependovirus-based vector containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, wherein said cells supply cap and/or rep function that the recombinant construct lacks; or (v) transfecting the susceptible cells with a Dependovirus-based vector lacking functioning cap and/or rep and plasmids for inserting exogenous DNA into the recombinant construct so that the exogenous DNA is expressed by the recombinant construct and for supplying rep and/or cap functions whereby transfection results in a Dependovirus-based vector containing or consisting essentially of the exogenous DNA including DNA for expression that lacks functioning cap and/or rep; and (b) incubating the infected or transfected cells, whereby there results infected or transfected cells and supernatant containing the Dependovirus-based vector lacking functioning cap and/or rep; (c) after incubating, extracting an aliquot from the supernatant; (d) filtering the aliquot, whereby the filtered aliquot contains and the method obtains a sample containing set amount of the Dependovirus-based vector relative to the type and amount of susceptible cells infected or transfected; and (e) optionally freezing the filtered aliquot, whereby the method optionally includes storing a sample containing set amount of the Dependovirus-based vector relative to the type and amount of susceptible cells infected or transfected.

In one aspect, the Dependovirus-based vector of the invention is derived from one or more Dependoviruses selected from one or more of: adeno associated virus (AAV), Adenovirus, parvovirus, Erythrovirus, Bocavirus and the like. In one aspect, the Dependovirus-based vector of the invention is derived from a recombinant adeno associated virus (rAAV).

The invention is further described by the following numbered paragraphs:

1. A method for obtaining and optionally storing a sample containing a set amount of rAAV comprising or consisting essentially of:

(a) creating infected or transfected cells by a process comprising or consisting essentially of one or more methods selected from:

(i) transfecting plasmid(s) containing or consisting essentially of exogenous DNA including DNA for expression into AAV-infected cells along with another helper plasmid that provides AAV rep and/or cap genes which are obligatory for replication and packaging of the rAAV; or

(ii) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, and helper virus wherein the rAAV lacks functioning cap and/or rep and the helper virus provides the cap and/or rev function that the rAAV lacks; or

(iii) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, and transfecting said cells with a plasmid supplying cap and/or rep function that the rAAV lacks; or

(iv) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, wherein said cells supply cap and/or rep function that the recombinant construct lacks; or

(v) transfecting the susceptible cells with an AAV lacking functioning cap and/or rep and plasmids for inserting exogenous DNA into the recombinant construct so that the exogenous DNA is expressed by the recombinant construct and for supplying rep and/or cap functions whereby transfection results in an rAAV containing or consisting essentially of the exogenous DNA including DNA for expression that lacks functioning cap and/or rep; and

(b) incubating the infected or transfected cells, whereby there results infected or transfected cells and supernatant containing the rAAV lacking functioning cap and/or rep;

(c) after incubating, extracting an aliquot from the supernatant;

(d) filtering the aliquot, whereby the filtered aliquot contains and the method obtains a sample containing set amount of the rAAV relative to the type and amount of susceptible cells infected or transfected; and

(e) optionally freezing the filtered aliquot,

whereby the method optionally includes storing a sample containing set amount of the rAAV relative to the type and amount of susceptible cells infected or transfected.

2. A method for screening rAAV comprising or consisting essentially of,

preparing the filtered aliquot or the stored filtered aliquot of paragraph 1,

if necessary, thawing the stored filtered aliquot,

contacting the filtered aliquot with cells, and

determining whether the exogenous DNA is expressed in an amount and/or duration sufficient for an intended use.

3. The method of paragraph 2 wherein the contacting of the filtered aliquot with cells comprises or consists essentially of transducing said cells.

4. The method of paragraph 3 wherein the contacting is for 5-6 days.

5. The method of paragraph 2 wherein the rAAV expresses a TALE and the contacting includes or consists essentially of detecting nuclease, activator or repressor activity.

6. The method of paragraph 2 wherein the rAAV expresses a LITE, and the contacting includes or consists essentially of inducing gene expression or subjecting the contacted cells to a suitable stimulus, and detecting whether a transcriptional effector has been induced.

7. The method of paragraph 6 wherein detecting whether a transcriptional effector has been induced includes or consists essentially of detecting a color change.

8. The method of paragraph 2 wherein the rAAV expresses a CRISPR system, and the contacting includes or consists essentially of detecting gene knockdown or other effects of the CRISPR system.

9. The method of paragraph 1 or 2 wherein the AAV is AAV1, AAV2, AAV5 or an AAV having a hybrid or mosaic AAV1, AAV2 and/or AAV5 capsid.

10. The method of paragraph 1 or 2 wherein the susceptible cells are 293FT cells.

11. The method of paragraph 10 wherein 2×10⁵ cells are transfected or infected.

12. The method of paragraph 11 wherein a 250 μL filtered aliquot contains the recombinant AAV at a concentration of about 5.6+/−0.24×10⁵.

13. The method of any one of paragraphs 1 or 2 including freezing the filtered aliquot.

14. The method of paragraph 13 wherein the filtered aliquot is frozen at about −80 C.

15. The method of any one of paragraphs 1 or 2 including adding a secretion enhancer to the cells before, during or after and within the incubating.

16. The method of paragraph 15 wherein the secretion enhancer is polyethylenimine (PEI).

17. A method of high-throughput screening of a sample comprising or consisting essentially of contacting the supernatant containing the rAAV lacking functioning cap and/or rep of any one of paragraphs 1-16 with the sample and determining whether the exogenous DNA of paragraph 1 is present in the sample.

18. The method of paragraph 17, wherein the supernatant is thawed from the filtered aliquot.

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention. 

What is claimed is:
 1. A method for obtaining and optionally storing a sample containing a set amount of rAAV comprising or consisting essentially of: (a) creating infected or transfected cells by a process comprising or consisting essentially of one or more methods selected from: (i) transfecting plasmid(s) containing or consisting essentially of exogenous DNA including DNA for expression into AAV-infected cells along with another helper plasmid that provides AAV rep and/or cap genes which are obligatory for replication and packaging of the rAAV; or (ii) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, and helper virus wherein the rAAV lacks functioning cap and/or rep and the helper virus provides the cap and/or rev function that the rAAV lacks; or (iii) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, and transfecting said cells with a plasmid supplying cap and/or rep function that the rAAV lacks; or (iv) infecting susceptible cells with a rAAV containing or consisting essentially of exogenous DNA including DNA for expression, wherein the recombinant construct lacks functioning cap and/or rep, wherein said cells supply cap and/or rep function that the recombinant construct lacks; or (v) transfecting the susceptible cells with an AAV lacking functioning cap and/or rep and plasmids for inserting exogenous DNA into the recombinant construct so that the exogenous DNA is expressed by the recombinant construct and for supplying rep and/or cap functions whereby transfection results in an rAAV containing or consisting essentially of the exogenous DNA including DNA for expression that lacks functioning cap and/or rep; and (b) incubating the infected or transfected cells, whereby there results infected or transfected cells and supernatant containing the rAAV lacking functioning cap and/or rep; (c) after incubating, extracting an aliquot from the supernatant; (d) filtering the aliquot, whereby the filtered aliquot contains and the method obtains a sample containing set amount of the rAAV relative to the type and amount of susceptible cells infected or transfected; and (e) optionally freezing the filtered aliquot, whereby the method optionally includes storing a sample containing set amount of the rAAV relative to the type and amount of susceptible cells infected or transfected.
 2. A method for screening rAAV comprising or consisting essentially of, preparing the filtered aliquot or the stored filtered aliquot of claim 1, if necessary, thawing the stored filtered aliquot, contacting the filtered aliquot with cells, and determining whether the exogenous DNA is expressed in an amount and/or duration sufficient for an intended use.
 3. The method of claim 2 wherein the contacting of the filtered aliquot with cells comprises or consists essentially of transducing said cells.
 4. The method of claim 3 wherein the contacting is for 5-6 days.
 5. The method of claim 2 wherein the rAAV expresses a TALE and the contacting includes or consists essentially of detecting nuclease, activator or repressor activity.
 6. The method of claim 2 wherein the rAAV expresses a LITE, and the contacting includes or consists essentially of inducing gene expression or subjecting the contacted cells to a suitable stimulus, and detecting whether a transcriptional effector has been induced.
 7. The method of claim 6 wherein detecting whether a transcriptional effector has been induced includes or consists essentially of detecting a color change.
 8. The method of claim 2 wherein the rAAV expresses a CRISPR system, and the contacting includes or consists essentially of detecting gene knockdown or other effects of the CRISPR system.
 9. The method of claim 1 wherein the AAV is AAV1, AAV2, AAV5 or an AAV having a hybrid or mosaic AAV1, AAV2 and/or AAV5 capsid.
 10. The method of claim 1 wherein the susceptible cells are 293FT cells.
 11. The method of claim 10 wherein 2×10⁵ cells are transfected or infected.
 12. The method of claim 11 wherein a 250 μL filtered aliquot contains the recombinant AAV at a concentration of about 5.6+/−0.24×10⁵.
 13. The method of claim 1 including freezing the filtered aliquot.
 14. The method of claim 13 wherein the filtered aliquot is frozen at about −80 C.
 15. The method of claim 1 including adding a secretion enhancer to the cells before, during or after and within the incubating.
 16. The method of claim 15 wherein the secretion enhancer is polyethylenimine (PEI).
 17. A method of high-throughput screening of a sample comprising or consisting essentially of contacting the supernatant containing the rAAV lacking functioning cap and/or rep of claim 1 with the sample and determining whether the exogenous DNA of claim 1 is present in the sample.
 18. The method of claim 17, wherein the supernatant is thawed from the filtered aliquot. 